MERS-CoV Vaccine

ABSTRACT

Disclosed herein is a vaccine comprising a Middle East Respiratory Syndrome coronavirus (MERS-CoV) antigen. The antigen can be a consensus antigen. The consensus antigen can be a consensus spike antigen. Also disclosed herein is a method of treating a subject in need thereof, by administering the vaccine to the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/039,672, filed May 26, 2016, now allowed, which is a U.S.national phase application filed under 35 U.S.C. § 371 claiming benefitto International Patent Application No. PCT/US2014/067537, filed Nov.26, 2014, which is entitled to priority under 35 U.S.C § 119(e) to U.S.Provisional Patent Application No. 61/910,153, filed Nov. 29, 2013, eachof which applications are incorporated by reference herein in theirentireties.

TECHNICAL FIELD

The present invention relates to a vaccine for Middle East RespiratorySyndrome coronavirus (MERS-CoV) and a method of administering thevaccine.

BACKGROUND

Coronaviruses (CoV) are a family of viruses that are common worldwideand cause a range of illnesses in humans from the common cold to severeacute respiratory syndrome (SARS). Coronaviruses can also cause a numberof diseases in animals. Human coronaviruses 229E, OC43, NL63, and HKU1are endemic in the human population.

In 2012, a novel coronavirus (nCoV) emerged in Saudi Arabia and is nowknown as Middle East Respiratory Syndrome coronavirus (MERS-CoV) (FIG.1). MERS-CoV can be classified as a beta coronavirus (FIG. 2, starredMERS-CoV strain HCoV-EMC/2012). Subsequent cases of MERS-CoV infectionhave been reported elsewhere in the Middle East (e.g., Qatar and Jordan)and more recently in Europe. Infection with MERS-CoV presented as severeacute respiratory illness with symptoms of fever, cough, and shortnessof breath. About half of reported cases of MERS-CoV infection haveresulted in death and a majority of reported cases have occurred inolder to middle age men. Only a small number of reported cases involvedsubjects with mild respiratory illness. Human to human transmission ofMERS-CoV is possible, but very limited at this time.

Accordingly, a need remains in the art for the development of a safe andeffective vaccine that is applicable to MERS-CoV, thereby providingprotection against and promoting survival of MERS-CoV infection.

SUMMARY

The present invention is directed to an immunogenic composition. In oneembodiment, the invention is directed to a vaccine comprising a nucleicacid molecule, wherein the nucleic acid molecule can comprise a nucleicacid sequence having at least about 90% identity over an entire lengthof the nucleic acid sequence set forth in SEQ ID NO:1 or the nucleicacid molecule can comprise a nucleic acid sequence having at least about90% identity over an entire length of the nucleic acid sequence setforth in SEQ ID NO:3.

The present invention is also directed to a vaccine comprising a nucleicacid molecule, wherein the nucleic acid molecule can encode a peptidecomprising an amino acid sequence having at least about 90% identityover an entire length of the amino acid sequence set forth in SEQ IDNO:2 or the nucleic acid molecule can encode a peptide comprising anamino acid sequence having at least about 90% identity over an entirelength of the amino acid sequence set forth in SEQ ID NO:4.

The present invention is further directed to a nucleic acid moleculecomprising the nucleic acid sequence set forth in SEQ ID NO:1.

The present invention is further directed to a nucleic acid moleculecomprising the nucleic acid sequence set forth in SEQ ID NO:3.

The present invention is further directed to a peptide comprising theamino acid sequence set forth in SEQ ID NO:2.

The present invention is further directed to a peptide comprising theamino acid sequence set forth in SEQ ID NO:4.

The present invention is further directed to a vaccine comprising anantigen, wherein the antigen is encoded by SEQ ID NO:1 or SEQ ID NO:3.

The present invention is also directed to a vaccine comprising apeptide, wherein the peptide can comprise an amino acid sequence havingat least about 90% identity over an entire length of the amino acidsequence set forth in SEQ ID NO:2 or the peptide can comprise an aminoacid sequence having at least about 90% identity over an entire lengthof the amino acid sequence set forth in SEQ ID NO:4.

The present invention is further directed to a method of inducing animmune response against a Middle East Respiratory Syndrome coronavirus(MERS-CoV) in a subject in need thereof. The method can compriseadministering one or more of the above vaccines, nucleic acid molecules,or peptides to the subject.

The present invention is further directed to a method of protecting asubject in need thereof from infection with a Middle East RespiratorySyndrome coronavirus (MERS-CoV). The method can comprise administeringone or more of the above vaccines, nucleic acid molecules, or peptidesto the subject.

The present invention is further directed to a method of treating asubject in need thereof against Middle East Respiratory Syndromecoronavirus (MERS-CoV). The method can comprise administering one ormore of the above vaccines, nucleic acid molecules, or peptides to thesubject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a timeline illustrating the emergence of MERS-CoV.

FIG. 2 shows a phylogenetic tree depicting the relationship between theindicated coronaviruses.

FIG. 3 shows a phylogenetic tree depicting the relationship between theindicated MERS-CoV and a MERS-CoV consensus spike antigen.

FIG. 4A shows a schematic illustrating the MERS-HCoV-WT construct; andFIG. 4B shows an image of a stained gel.

FIG. 5 shows a graph generated by the web-based software Phobius.

FIG. 6 shows a schematic generated by the web-based software InterProScan.

FIG. 7A shows a schematic illustrating the MERS-HCoV-ΔCD construct; andFIG. 7B shows an image of a stained gel.

FIG. 8A shows a nucleotide sequence encoding a MERS-CoV consensus spikeantigen; FIG. 8B shows the amino acid sequence of the MERS-CoV consensusspike antigen;

FIG. 8C shows a nucleotide sequence encoding a MERS-CoV consensus spikeantigen that lacks the cytoplasmic domain (MERS-CoV consensus spikeantigen ΔCD); and FIG. 8D shows the amino acid sequence of the MERS-CoVconsensus spike antigen ΔCD.

FIG. 9 shows an immunoblot.

FIG. 10 shows a schematic illustrating an immunization regimen.

FIG. 11A and FIG. 11B show graphs plotting bleed vs. OD450.

FIG. 12 shows a matrix of peptide pools.

FIG. 13 shows a graph plotting peptide pool vs. spot forming unit per10⁶ cells (SFU/10⁶ cells).

FIG. 14 shows a graph plotting peptide pool vs. spot forming unit per10⁶ cells (SFU/10⁶ cells).

FIG. 15 shows a graph plotting immunization group vs. spot forming unitper 10⁶ cells (SFU/10⁶ cells).

FIG. 16A shows a graph plotting immunization group vs. percentCD3⁺CD4⁺IFN-γ⁺ T cells; FIG. 16B shows a graph plotting immunizationgroup vs. percent CD3⁺CD4⁺TNF-α⁺ T cells; FIG. 16C shows a graphplotting immunization group vs. percent CD3⁺CD4⁺IL-2⁺ T cells; and FIG.16D shows a graph plotting immunization group vs. percentCD3⁺CD4⁺IFN-γ⁺TNF-α⁺ T cells.

FIG. 17A shows a graph plotting immunization group vs. percentCD3⁺CD8⁺IFN-γ⁺ T cells; FIG. 17B shows a graph plotting immunizationgroup vs. percent CD3⁺CD8⁺TNF-α⁺ T cells; FIG. 17C shows a graphplotting immunization group vs. percent CD3⁺CD8⁺IL-2⁺ T cells; and FIG.17D shows a graph plotting immunization group vs. percentCD3⁺CD8⁺IFN-γ⁺TNF-α⁺ T cells.

FIG. 18A shows the antibody response after vaccination with pVax1 DNAvector (negative control), consensus MERS-Spike DNA vaccine (MERS-S), orconsensus MERS-Spike-ΔCD DNA vaccine (MERS-S-CD). The antibody responsewas measured by ELISA. In FIG. 18B, the antibody response over timeafter vaccination with consensus MERS-Spike DNA vaccine (MERS-S) isshown. In FIG. 18C, the neutralizing antibody titer at day 35 is shownafter vaccination with pVax1 DNA vector (negative control), MERS-S DNAvaccine, or MERS-S-CD DNA vaccine. Mouse sera were serially diluted inMEM and incubate with 50 ul of DMEM containing 100 infectiousHCoV-EMC/2012 (Human Coronavirus Erasmus Medical Center/2012) particlesper well at 37° C. After 90 min, the virus-serum mixture was add to amonolayer of Vero cells (100,000 cells/per well) in a 96-well flatbottom plate and incubate for 5 days at 37° C. in a 5% CO₂ incubator.The titer of neutralizing antibody for each sample is reported as thehighest dilution with which less than 50% of the cells show CPE. Valuesare reported as reciprocal dilutions. All the samples were run induplicate so the final result was taken as the average of the two. Thepercent neutralization was calculated as follows: Percentneutralization={1-PFU mAb of interest (each concentration)/Mean PFUnegative control (all concentrations)}.

FIG. 19, comprising FIG. 19A through FIG. 19D, shows the constructionand characterization of MERS-HCoV Spike DNA vaccine. FIG. 19A providesdendograms showing the phylogenetic relationships between the MERS-HCoVviral isolates and Spike vaccine strains at the amino acid level. Arooted neighbor joining tree was generated from amino acid sequencealignments of Spike protein with the consensus vaccine displayed as (★)and (

) denotes the MERS-viral strain used to test the Nab assay. The shadedarea represents reported cases from the 2012-2013 outbreaks. Parenthesisindicated the specific clades. The tree was drawn to scale (scale bar,0.1 percent difference). FIG. 19B provides a schematic diagram ofSpike-Wt gene inserts used in codon optimized DNA vaccines. Spike-Wt andSpikeΔCD-DNA vaccines were constructed. Different Spike protein domainswere indicated. FIG. 19C shows that expression of protein was detectedby Western blot. The expression of Spike-Wt and SpikeΔCD-Spike proteinwas analyzed two days post transfection by Western blotting using serafrom the MERS-Spike-Wt DNA vaccinated mice at 1:100 dilutions. DNAconstruct transfected and amount of cell lysates loaded as indicated.Arrow indicated the Spike protein. FIG. 19D shows an immune fluorescenceassay in Vero cells, which showed the Spike-Wt and SpikeΔCD proteinexpression using the sera (1:100) from DNA immunized mice.

FIG. 20, comprising FIG. 20A through FIG. 20C, shows the constructionand characterization of MERS-Spike pseudovirus. FIG. 20A provides aschematic representation of MERS-HCoV-Spike pesudovirus. FIG. 20B showsthe determination of viral infectivity: Vero cells were incubated withMERS-Spike pseudo-typed with Luciferase reporter backbone. Luciferasereporter activity was then assayed at the indicated times to produce atime course of expression. Curves were fitted to the data by non-linearregression analysis and the best fit was a straight line. FIG. 20C showsthe detection of MERS-HCoV-Spike pseudovirus infectivity in differentcell lines. MERS-Spike pseudoviruses and the negative control VSV-Gvirus were used for the infection. The data were expressed as meanrelative luciferase units (RLU)±standard deviation (SD) of 3 parallelwells in 96-well culture plates. The experiment was repeated threetimes, and similar results were obtained.

FIG. 21, comprising FIG. 21A through FIG. 21D, shows the functionalprofile of cellular immune responses elicited by MERS-HCoV vaccines.FIG. 21A shows that groups of C57/BL6 mice (n=9/group) were immunizedthree times, each 2 weeks apart with 25 μg of Spike-Wt and Spike ΔCD DNAvaccine as indicated. Samples were collected a week after the thirdimmunization. FIG. 21B shows that spike-specific T-lymphocyte responseswere assessed by IFN-γ ELISpot assays using 6 peptide pools covering theSpike protein. Values represented mean responses in each group one weekafter the third immunization. Error bars indicated standard errors. FIG.21C and FIG. 21D show the characterization of MERS-Spike-specificdominant epitopes. Secretion of IFN-γ by splenocytes fromMERS-Spike-specific immune responses using matrix pooling of peptides.The results were expressed as the number of IFN-γ secreting cells/10⁶spleen cells (mean±SD for three mice per group) after subtracting valueswithout peptide stimulation. Similar results were obtained in twoseparate experiments.

FIG. 22, comprising FIG. 22A through FIG. 22E, shows systemicanti-MERS-HCoV IgG levels after DNA immunization. FIG. 22A shows theserum anti-IgG responses in mice measured by ELISA with as indicatedserum dilution against MERS-Spike as coating antigen. Pooled serum fromindividual groups of mice (one week after the 3rd immunization) wasserially diluted and MERS-Spike-specific total IgG was measured byELISA. Each curve represented the OD value of each group of 9 micereceiving Spike-Wt DNA vaccine, SpikeΔCD-Spike DNA vaccine or empty DNAvector as indicated. Each data point was the average absorbance fromnine mice. FIG. 22B shows the endpoint titer for the Spike-Wt immunizedmice sera were calculated at the indicated time points. FIG. 22C shows awestern blot analysis of immunized sera binding to recombinant Spikeprotein at indicated concentrations (μg). Pooled sera from the Spike-Wtimmunized mice was used as the primary antibody at 1:100 dilution. FIG.22D shows the neutralizing antibody responses detected by the viralinfection assay. Sera, at one week after the third immunization, werecollected from the immunized mice. Neutralizing antibody titers weremeasured at 50% inhibition (IC₅₀) of virus infection to target cells.Data shown were the geometric mean titers of each group with standarddeviations. The statistical differences between each testing group weredetermined and p values were indicated. FIG. 22E shows theneutralization of the infectivity of MERS-Spike-pseudotyped viruses byantisera obtained from mice after receiving three injections of Spike-WtDNA or pVax1 DNA. The IC₅₀ was defined as the reciprocal of theantiserum dilution at which virus entry is 50% inhibited (dashed line).Data from 4 mice per each group were shown.

FIG. 23, comprising FIG. 23A through FIG. 23C, shows the analysis ofinterferon-gamma (IFN-γ) producing cells induced by MERS-Spike DNAvaccine in non-human primates (NHP). FIG. 23A shows the study design,vaccine and challenge regimen in rhesus monkeys. Three groups (low, highand control) of rhesus monkeys (n=4/each group) were immunized withMERS-Spike DNA at 0, 3, and 6 weeks (wks) as indicated. IFN-γ ELISpot,intracellular cytokine staining and antibody binding and Nab assay wereperformed from the 3rd immunization samples. Biopsies were performedfollowing MERS-Challenge. FIG. 23B shows an ELISPOT analysis of cellsfrom MERS-Spike DNA-immunized monkeys. PBMCs were isolated from each ofDNA-immunized monkeys and were used for ELISPOT assay to detectIFN-γ-producing cells responding to pools of MERS-Spike peptides for 24hours as described in Example 9. Frequencies of MERS-Spike specificIFN-γ-secreting cells/10⁶ PBMCs were determined by ELISpot assay.Results were presented as mean±SEM. FIG. 23C shows the frequency ofvaccine induced cytokine (IFN-γ, IL-2, or TNF-α) producing CD4⁺ and CD8⁺T cells. Macaques PBMC's (n=4) were isolated after the final DNAimmunization and were stimulated with pooled MERS-Spike peptide ex-vivo.Cells were stained for intracellular production of IFN-γ, TNF-α, andIL-2, and then analyzed by multi parameter flow cytometry.

FIG. 24, comprising FIG. 24A through FIG. 24D, shows antibody bindingtiters and neutralizing antibody (Nab) response following MERS-Spike DNAvaccinations. FIG. 24A and FIG. 24B show MERS-Specific antibodies inserum binding and IgG specific end point titers at various timespost-immunization. Serum was collected after each vaccination andendpoint titers calculated by ELISA. FIG. 24C and FIG. 24D show theeffect of DNA vaccinations and characterization of the neutralizingantibody response. Blood samples were obtained from the animals prior toeach immunization and two weeks after the final vaccination. The serawere tested for neutralizing antibody. Neutralization with live virus(FIG. 24C) or MERS-pseudovirus (FIG. 24D). Serially diluted immunizedsera were tested with MERS-virus. For the pseudovirus neutralizationassay, an anti-CHIKV monkey serum was used as a negative antibodycontrol, and VSV-G pseudotyped virus was used as pseudovirus control forneutralization specificity. Samples were tested by PRNT50 assay withMERS virus.

FIG. 25, comprising FIG. 25A through FIG. 25D, shows the functionalprofile of CD4⁺ and CD8⁺ T cell responses elicited by MERS-Spikevaccine. The cytokine profiles of specific CD4⁺ T cells and CD8⁺ T cellsinduced after vaccination are shown in FIG. 25A and FIG. 25B,respectively. Mouse splenocytes (n=3) were isolated one week after thefinal DNA immunization and were stimulated with pooled MERS-Spikepeptide ex-vivo. Cells were stained for intracellular production ofIFN-γ, TNF-α, and IL-2, and then analyzed by FACS. FIG. 25A and FIG. 25Bprovide scatter plots depicting MERS-specific CD4⁺ T and CD8⁺ T cellsreleasing IFN-γ, TNF-α, IL-2 and dual IFN-γ/TNF-α cytokines. FIG. 25Cand FIG. 25D provide column graph shows multifunctional subpopulationsof single-, double- and triple-positive CD4⁺ and CD8⁺ T cells releasingthe cytokines IFN-γ, TNF-α, and IL-2. The pie charts show the proportionof each cytokine subpopulation. Data represented mean±SEM of three miceper group.

FIG. 26, comprising FIG. 26A through FIG. 26B, shows the inhibition ofapoptosis induced by MERS-S pseudoviruses by Spike DNA immunized mouseimmune sera. FIG. 26A shows MERS-HCoV-Spike mouse sera neutralized MERSin vitro and blocks syncytia formation. Vero cells, which wereuninfected, represented a negative control, whereas the positive controlrepresented those cells infected with MERS-HCoV pseudovirus. Arrowindicated the syncytia formation. Syncytia formation was observed underthe microscope 36 hours post infection. FIG. 26B shows MERS-Spikepseudoviruses pre-incubated in the presence of either pVax-1 orMERS-Spike immunized sera at 1:100 dilutions and added to the Verocells. Two days post infection, the percentage of cell death wasmeasured by the Annexin V/PI staining. Bar graph indicated the infectionof MERS-pseudovirus values from the FACS data for a single experimentcarried out in triplicate. Similar results were obtained in threeindependent experiments.

FIG. 27, comprising FIG. 27A through FIG. 27B, shows the antibodyresponse after vaccination with MERS-Spike DNA vaccine in combinationwith electroporation. Sera from monkeys vaccinated with pVax1,pMERS-Spike (0.5 mg/animal; low dose), or pMERS-Spike (2 mg/animal; highdose) were collected two weeks after the third immunization. These serawere tested for antibodies that bound MERS-Spike protein. The results ofthis testing are shown in FIG. 27A and FIG. 27B.

FIG. 28, comprising FIG. 28A through FIG. 28B, shows results from MERSchallenge post-vaccination and pathobiology. FIG. 28A shows the meanviral load determined by qRT-PCR from individual tissues collected atnecropsy. FIG. 28B shows the mean viral load of all lung lobes combinedis indicated in the inset. Log TCID50 eq/g, log TCID50 equivalents pergram tissue; one sample per tissue per animal from three animals pergroup were analyzed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a vaccine comprising a Middle EastRespiratory Syndrome coronavirus (MERS-CoV) antigen. MERS-CoV is a newand highly pathogenic virus, only emerging in 2012, and thus, thevaccine described herein is one of the first vaccines to targetMERS-CoV. Accordingly, the vaccine provides a treatment for this new andpathogenic virus, for which prior treatment did not exist and potentialfor a pandemic remains.

The MERS-CoV antigen can be a MERS-CoV consensus spike antigen. TheMERS-CoV consensus spike antigen can be derived from the sequences ofspike antigens from strains of MERS-CoV, and thus, the MERS-CoVconsensus spike antigen is unique. The MERS-CoV consensus spike antigencan lack a cytoplasmic domain. Accordingly, the vaccine of the presentinvention is widely applicable to multiple strains of MERS-CoV becauseof the unique sequences of the MERS-CoV consensus spike antigen. Theseunique sequences allow the vaccine to be universally protective againstmultiple strains of MERS-CoV, including genetically diverse variants ofMERS-CoV.

The vaccine can be used to protect against and treat any number ofstrains of MERS-CoV. The vaccine can elicit both humoral and cellularimmune responses that target the MERS-CoV spike antigen. The vaccine canelicit neutralizing antibodies and immunoglobulin G (IgG) antibodiesthat are reactive with the MERS-CoV spike antigen. The vaccine can alsoelicit CD8⁺ and CD4⁺ T cell responses that are reactive to the MERS-CoVspike antigen and produce interferon-gamma (IFN-γ), tumor necrosisfactor alpha (TNF-α), and interleukin-2 (IL-2).

1. DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the contextclearly dictates otherwise. The present disclosure also contemplatesother embodiments “comprising,” “consisting of” and “consistingessentially of,” the embodiments or elements presented herein, whetherexplicitly set forth or not.

“Adjuvant” as used herein means any molecule added to the vaccinedescribed herein to enhance the immunogenicity of the antigen.

“Antibody” as used herein means an antibody of classes IgG, IgM, IgA,IgD or IgE, or fragments, fragments or derivatives thereof, includingFab, F(ab′)₂, Fd, and single chain antibodies, diabodies, bispecificantibodies, bifunctional antibodies and derivatives thereof. Theantibody can be an antibody isolated from the serum sample of mammal, apolyclonal antibody, affinity purified antibody, or mixtures thereofwhich exhibits sufficient binding specificity to a desired epitope or asequence derived therefrom.

“Coding sequence” or “encoding nucleic acid” as used herein means thenucleic acids (RNA or DNA molecule) that comprise a nucleotide sequencewhich encodes a protein. The coding sequence can further includeinitiation and termination signals operably linked to regulatoryelements including a promoter and polyadenylation signal capable ofdirecting expression in the cells of an individual or mammal to whichthe nucleic acid is administered.

“Complement” or “complementary” as used herein means Watson-Crick (e.g.,A-T/U and C-G) or Hoogsteen base pairing between nucleotides ornucleotide analogs of nucleic acid molecules.

“Consensus” or “Consensus Sequence” as used herein may mean a syntheticnucleic acid sequence, or corresponding polypeptide sequence,constructed based on analysis of an alignment of multiple subtypes of aparticular antigen. The sequence may be used to induce broad immunityagainst multiple subtypes, serotypes, or strains of a particularantigen. Synthetic antigens, such as fusion proteins, may be manipulatedto generate consensus sequences (or consensus antigens).

“Electroporation,” “electro-permeabilization,” or “electro-kineticenhancement” (“EP”) as used interchangeably herein means the use of atransmembrane electric field pulse to induce microscopic pathways(pores) in a bio-membrane; their presence allows biomolecules such asplasmids, oligonucleotides, siRNA, drugs, ions, and water to pass fromone side of the cellular membrane to the other.

“Fragment” as used herein means a nucleic acid sequence or a portionthereof that encodes a polypeptide capable of eliciting an immuneresponse in a mammal. The fragments can be DNA fragments selected fromat least one of the various nucleotide sequences that encode proteinfragments set forth below.

“Fragment” or “immunogenic fragment” with respect to polypeptidesequences means a polypeptide capable of eliciting an immune response ina mammal that cross reacts with a full length wild type strain MERS-CoVantigen. Fragments of consensus proteins can comprise at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% or at least 95% of a consensusprotein. In some embodiments, fragments of consensus proteins cancomprise at least 20 amino acids or more, at least 30 amino acids ormore, at least 40 amino acids or more, at least 50 amino acids or more,at least 60 amino acids or more, at least 70 amino acids or more, atleast 80 amino acids or more, at least 90 amino acids or more, at least100 amino acids or more, at least 110 amino acids or more, at least 120amino acids or more, at least 130 amino acids or more, at least 140amino acids or more, at least 150 amino acids or more, at least 160amino acids or more, at least 170 amino acids or more, at least 180amino acids or more, at least 190 amino acids or more, at least 200amino acids or more, at least 210 amino acids or more, at least 220amino acids or more, at least 230 amino acids or more, or at least 240amino acids or more of a consensus protein.

As used herein, the term “genetic construct” refers to the DNA or RNAmolecules that comprise a nucleotide sequence which encodes a protein.The coding sequence includes initiation and termination signals operablylinked to regulatory elements including a promoter and polyadenylationsignal capable of directing expression in the cells of the individual towhom the nucleic acid molecule is administered. As used herein, the term“expressible form” refers to gene constructs that contain the necessaryregulatory elements operable linked to a coding sequence that encodes aprotein such that when present in the cell of the individual, the codingsequence will be expressed.

“Identical” or “identity” as used herein in the context of two or morenucleic acids or polypeptide sequences, means that the sequences have aspecified percentage of residues that are the same over a specifiedregion. The percentage can be calculated by optimally aligning the twosequences, comparing the two sequences over the specified region,determining the number of positions at which the identical residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the specified region, and multiplying the result by 100 toyield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of single sequence are included in thedenominator but not the numerator of the calculation. When comparing DNAand RNA, thymine (T) and uracil (U) can be considered equivalent.Identity can be performed manually or by using a computer sequencealgorithm such as BLAST or BLAST 2.0.

“Immune response” as used herein means the activation of a host's immunesystem, e.g., that of a mammal, in response to the introduction ofantigen. The immune response can be in the form of a cellular or humoralresponse, or both.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” as used hereinmeans at least two nucleotides covalently linked together. The depictionof a single strand also defines the sequence of the complementarystrand. Thus, a nucleic acid also encompasses the complementary strandof a depicted single strand. Many variants of a nucleic acid can be usedfor the same purpose as a given nucleic acid. Thus, a nucleic acid alsoencompasses substantially identical nucleic acids and complementsthereof. A single strand provides a probe that can hybridize to a targetsequence under stringent hybridization conditions. Thus, a nucleic acidalso encompasses a probe that hybridizes under stringent hybridizationconditions.

Nucleic acids can be single stranded or double stranded, or can containportions of both double stranded and single stranded sequence. Thenucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid can contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids can be obtained by chemical synthesismethods or by recombinant methods.

“Operably linked” as used herein means that expression of a gene isunder the control of a promoter with which it is spatially connected. Apromoter can be positioned 5′ (upstream) or 3′ (downstream) of a geneunder its control. The distance between the promoter and a gene can beapproximately the same as the distance between that promoter and thegene it controls in the gene from which the promoter is derived. As isknown in the art, variation in this distance can be accommodated withoutloss of promoter function.

A “peptide,” “protein,” or “polypeptide” as used herein can mean alinked sequence of amino acids and can be natural, synthetic, or amodification or combination of natural and synthetic.

“Promoter” as used herein means a synthetic or naturally-derivedmolecule which is capable of conferring, activating or enhancingexpression of a nucleic acid in a cell. A promoter can comprise one ormore specific transcriptional regulatory sequences to further enhanceexpression and/or to alter the spatial expression and/or temporalexpression of same. A promoter can also comprise distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A promoter can bederived from sources including viral, bacterial, fungal, plants,insects, and animals. A promoter can regulate the expression of a genecomponent constitutively or differentially with respect to cell, thetissue or organ in which expression occurs or, with respect to thedevelopmental stage at which expression occurs, or in response toexternal stimuli such as physiological stresses, pathogens, metal ions,or inducing agents. Representative examples of promoters include thebacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lacoperator-promoter, tac promoter, SV40 late promoter, SV40 earlypromoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40late promoter and the CMV IE promoter.

“Signal peptide” and “leader sequence” are used interchangeably hereinand refer to an amino acid sequence that can be linked at the aminoterminus of a MERS-CoV protein set forth herein. Signal peptides/leadersequences typically direct localization of a protein. Signalpeptides/leader sequences used herein preferably facilitate secretion ofthe protein from the cell in which it is produced. Signalpeptides/leader sequences are often cleaved from the remainder of theprotein, often referred to as the mature protein, upon secretion fromthe cell. Signal peptides/leader sequences are linked at the N terminusof the protein.

“Subject” as used herein can mean a mammal that wants to or is in needof being immunized with the herein described vaccine. The mammal can bea human, chimpanzee, dog, cat, horse, cow, mouse, or rat.

“Substantially identical” as used herein can mean that a first andsecond amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1100 or more amino acids. Substantiallyidentical can also mean that a first nucleic acid sequence and a secondnucleic acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1100 or more nucleotides.

“Treatment” or “treating,” as used herein can mean protecting of ananimal from a disease through means of preventing, suppressing,repressing, or completely eliminating the disease. Preventing thedisease involves administering a vaccine of the present invention to ananimal prior to onset of the disease. Suppressing the disease involvesadministering a vaccine of the present invention to an animal afterinduction of the disease but before its clinical appearance. Repressingthe disease involves administering a vaccine of the present invention toan animal after clinical appearance of the disease.

“Variant” used herein with respect to a nucleic acid means (i) a portionor fragment of a referenced nucleotide sequence; (ii) the complement ofa referenced nucleotide sequence or portion thereof; (iii) a nucleicacid that is substantially identical to a referenced nucleic acid or thecomplement thereof; or (iv) a nucleic acid that hybridizes understringent conditions to the referenced nucleic acid, complement thereof,or a sequences substantially identical thereto.

Variant can further be defined as a peptide or polypeptide that differsin amino acid sequence by the insertion, deletion, or conservativesubstitution of amino acids, but retain at least one biologicalactivity. Representative examples of “biological activity” include theability to be bound by a specific antibody or to promote an immuneresponse. Variant can also mean a protein with an amino acid sequencethat is substantially identical to a referenced protein with an aminoacid sequence that retains at least one biological activity. Aconservative substitution of an amino acid, i.e., replacing an aminoacid with a different amino acid of similar properties (e.g.,hydrophilicity, degree and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art. Kyte et al., J. Mol. Biol.157:105-132 (1982). The hydropathic index of an amino acid is based on aconsideration of its hydrophobicity and charge. It is known in the artthat amino acids of similar hydropathic indexes can be substituted andstill retain protein function. In one aspect, amino acids havinghydropathic indexes of ±2 are substituted. The hydrophilicity of aminoacids can also be used to reveal substitutions that would result inproteins retaining biological function. A consideration of thehydrophilicity of amino acids in the context of a peptide permitscalculation of the greatest local average hydrophilicity of thatpeptide, a useful measure that has been reported to correlate well withantigenicity and immunogenicity. Substitution of amino acids havingsimilar hydrophilicity values can result in peptides retainingbiological activity, for example immunogenicity, as is understood in theart. Substitutions can be performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hydrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

A variant may be a nucleic acid sequence that is substantially identicalover the full length of the full gene sequence or a fragment thereof.The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical over the full length of the gene sequence or a fragmentthereof. A variant may be an amino acid sequence that is substantiallyidentical over the full length of the amino acid sequence or fragmentthereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical over the full length of the amino acid sequence or afragment thereof.

“Vector” as used herein means a nucleic acid sequence containing anorigin of replication. A vector can be a viral vector, bacteriophage,bacterial artificial chromosome or yeast artificial chromosome. A vectorcan be a DNA or RNA vector. A vector can be a self-replicatingextrachromosomal vector, and preferably, is a DNA plasmid.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

2. VACCINE

Provided herein are immunogenic compositions, such as vaccines,comprising a Middle East Respiratory Syndrome coronavirus (MERS-CoV)antigen, a fragment thereof, a variant thereof, or a combinationthereof. The vaccine can be used to protect against any number ofstrains of MERS-CoV, thereby treating, preventing, and/or protectingagainst MERS-CoV based pathologies. The vaccine can significantly inducean immune response of a subject administered the vaccine, therebyprotecting against and treating MERS-CoV infection.

The vaccine can be a DNA vaccine, a peptide vaccine, or a combinationDNA and peptide vaccine. The DNA vaccine can include a nucleic acidsequence encoding the MERS-CoV antigen. The nucleic acid sequence can beDNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combinationthereof. The nucleic acid sequence can also include additional sequencesthat encode linker, leader, or tag sequences that are linked to theMERS-CoV antigen by a peptide bond. The peptide vaccine can include aMERS-CoV antigenic peptide, a MERS-CoV antigenic protein, a variantthereof, a fragment thereof, or a combination thereof. The combinationDNA and peptide vaccine can include the above described nucleic acidsequence encoding the MERS-CoV antigen and the MERS-CoV antigenicpeptide or protein, in which the MERS-CoV antigenic peptide or proteinand the encoded MERS-CoV antigen have the same amino acid sequence.

The vaccine can induce a humoral immune response in the subjectadministered the vaccine. The induced humoral immune response can bespecific for the MERS-CoV antigen. The induced humoral immune responsecan be reactive with the MERS-CoV antigen. The humoral immune responsecan be induced in the subject administered the vaccine by about 1.5-foldto about 16-fold, about 2-fold to about 12-fold, or about 3-fold toabout 10-fold. The humoral immune response can be induced in the subjectadministered the vaccine by at least about 1.5-fold, at least about2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at leastabout 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, atleast about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold,at least about 6.5-fold, at least about 7.0-fold, at least about7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at leastabout 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, atleast about 10.5-fold, at least about 11.0-fold, at least about11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at leastabout 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, atleast about 14.5-fold, at least about 15.0-fold, at least about15.5-fold, or at least about 16.0-fold.

The humoral immune response induced by the vaccine can include anincreased level of neutralizing antibodies associated with the subjectadministered the vaccine as compared to a subject not administered thevaccine. The neutralizing antibodies can be specific for the MERS-CoVantigen. The neutralizing antibodies can be reactive with the MERS-CoVantigen. The neutralizing antibodies can provide protection againstand/or treatment of MERS-CoV infection and its associated pathologies inthe subject administered the vaccine.

The humoral immune response induced by the vaccine can include anincreased level of IgG antibodies associated with the subjectadministered the vaccine as compared to a subject not administered thevaccine. These IgG antibodies can be specific for the MERS-CoV antigen.These IgG antibodies can be reactive with the MERS-CoV antigen.Preferably, the humoral response is cross-reactive against two or morestrains of the MERS-CoV. The level of IgG antibody associated with thesubject administered the vaccine can be increased by about 1.5-fold toabout 16-fold, about 2-fold to about 12-fold, or about 3-fold to about10-fold as compared to the subject not administered the vaccine. Thelevel of IgG antibody associated with the subject administered thevaccine can be increased by at least about 1.5-fold, at least about2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at leastabout 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, atleast about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold,at least about 6.5-fold, at least about 7.0-fold, at least about7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at leastabout 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, atleast about 10.5-fold, at least about 11.0-fold, at least about11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at leastabout 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, atleast about 14.5-fold, at least about 15.0-fold, at least about15.5-fold, or at least about 16.0-fold as compared to the subject notadministered the vaccine.

The vaccine can induce a cellular immune response in the subjectadministered the vaccine. The induced cellular immune response can bespecific for the MERS-CoV antigen. The induced cellular immune responsecan be reactive to the MERS-CoV antigen. Preferably, the cellularresponse is cross-reactive against two or more strains of the MERS-CoV.The induced cellular immune response can include eliciting a CD8⁺ T cellresponse. The elicited CD8⁺ T cell response can be reactive with theMERS-CoV antigen. The elicited CD8⁺ T cell response can bepolyfunctional. The induced cellular immune response can includeeliciting a CD8⁺ T cell response, in which the CD8⁺ T cells produceinterferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α),interleukin-2 (IL-2), or a combination of IFN-γ and TNF-α.

The induced cellular immune response can include an increased CD8⁺ Tcell response associated with the subject administered the vaccine ascompared to the subject not administered the vaccine. The CD8⁺ T cellresponse associated with the subject administered the vaccine can beincreased by about 2-fold to about 30-fold, about 3-fold to about25-fold, or about 4-fold to about 20-fold as compared to the subject notadministered the vaccine. The CD8⁺ T cell response associated with thesubject administered the vaccine can be increased by at least about1.5-fold, at least about 2.0-fold, at least about 3.0-fold, at leastabout 4.0-fold, at least about 5.0-fold, at least about 6.0-fold, atleast about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold,at least about 8.0-fold, at least about 8.5-fold, at least about9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at leastabout 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, atleast about 12.0-fold, at least about 12.5-fold, at least about13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at leastabout 14.5-fold, at least about 15.0-fold, at least about 16.0-fold, atleast about 17.0-fold, at least about 18.0-fold, at least about19.0-fold, at least about 20.0-fold, at least about 21.0-fold, at leastabout 22.0-fold, at least about 23.0-fold, at least about 24.0-fold, atleast about 25.0-fold, at least about 26.0-fold, at least about27.0-fold, at least about 28.0-fold, at least about 29.0-fold, or atleast about 30.0-fold as compared to the subject not administered thevaccine.

The induced cellular immune response can include an increased frequencyof CD3⁺CD8⁺ T cells that produce IFN-γ. The frequency of CD3⁺CD8⁺IFN-γ⁺T cells associated with the subject administered the vaccine can beincreased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold,15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold as compared tothe subject not administered the vaccine.

The induced cellular immune response can include an increased frequencyof CD3⁺CD8⁺ T cells that produce TNF-α. The frequency of CD3⁺CD8⁺TNF-α⁺T cells associated with the subject administered the vaccine can beincreased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, or 14-foldas compared to the subject not administered the vaccine.

The induced cellular immune response can include an increased frequencyof CD3⁺CD8⁺ T cells that produce IL-2. The frequency of CD3⁺CD8⁺IL-2⁺ Tcells associated with the subject administered the vaccine can beincreased by at least about 0.5-fold, 1.0-fold, 1.5-fold, 2.0-fold,2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, or 5.0-fold ascompared to the subject not administered the vaccine.

The induced cellular immune response can include an increased frequencyof CD3⁺CD8⁺ T cells that produce both IFN-γ and TNF-α. The frequency ofCD3⁺CD8⁺IFN-γ⁺TNF-α⁺ T cells associated with the subject administeredthe vaccine can be increased by at least about 25-fold, 30-fold,35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold,75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 110-fold,120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, or 180-foldas compared to the subject not administered the vaccine.

The cellular immune response induced by the vaccine can includeeliciting a CD4⁺ T cell response. The elicited CD4⁺ T cell response canbe reactive with the MERS-CoV antigen. The elicited CD4⁺ T cell responsecan be polyfunctional. The induced cellular immune response can includeeliciting a CD4⁺ T cell response, in which the CD4⁺ T cells produceIFN-γ, TNF-α, IL-2, or a combination of IFN-γ and TNF-α.

The induced cellular immune response can include an increased frequencyof CD3⁺CD4⁺ T cells that produce IFN-γ. The frequency of CD3⁺CD4⁺IFN-γ⁺T cells associated with the subject administered the vaccine can beincreased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold,15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold as compared tothe subject not administered the vaccine.

The induced cellular immune response can include an increased frequencyof CD3⁺CD4⁺ T cells that produce TNF-α. The frequency of CD3⁺CD4⁺TNF-α⁺T cells associated with the subject administered the vaccine can beincreased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold,15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, or22-fold as compared to the subject not administered the vaccine.

The induced cellular immune response can include an increased frequencyof CD3⁺CD4⁺ T cells that produce IL-2. The frequency of CD3⁺CD4⁺IL-2⁺ Tcells associated with the subject administered the vaccine can beincreased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold,15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold,23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold,31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold,39-fold, 40-fold, 45-fold, 50-fold, 55-fold, or 60-fold as compared tothe subject not administered the vaccine.

The induced cellular immune response can include an increased frequencyof CD3⁺CD4⁺ T cells that produce both IFN-γ and TNF-α. The frequency ofCD3⁺CD4⁺IFN-γ⁺TNF-α⁺ associated with the subject administered thevaccine can be increased by at least about 2-fold, 2.5-fold, 3.0-fold,3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold,7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10.0-fold,10.5-fold, 11.0-fold, 11.5-fold, 12.0-fold, 12.5-fold, 13.0-fold,13.5-fold, 14.0-fold, 14.5-fold, 15.0-fold, 15.5-fold, 16.0-fold,16.5-fold, 17.0-fold, 17.5-fold, 18.0-fold, 18.5-fold, 19.0-fold,19.5-fold, 20.0-fold, 21-fold, 22-fold, 23-fold 24-fold, 25-fold,26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold,34-fold, or 35-fold as compared to the subject not administered thevaccine.

The vaccine of the present invention can have features required ofeffective vaccines such as being safe so the vaccine itself does notcause illness or death; is protective against illness resulting fromexposure to live pathogens such as viruses or bacteria; inducesneutralizing antibody to prevent invention of cells; induces protectiveT cells against intracellular pathogens; and provides ease ofadministration, few side effects, biological stability, and low cost perdose.

The vaccine can further induce an immune response when administered todifferent tissues such as the muscle or skin. The vaccine can furtherinduce an immune response when administered via electroporation, orinjection, or subcutaneously, or intramuscularly.

a. Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Antigen

As described above, the vaccine comprises a MERS-CoV antigen, a fragmentthereof, a variant thereof, or a combination thereof. Coronaviruses,including MERS-CoV, are encapsulated by a membrane and have a type 1membrane glycoprotein known as spike (S) protein, which forms protrudingspikes on the surface of the coronavirus. The spike protein facilitatesbinding of the coronavirus to proteins located on the surface of a cell,for example, the metalloprotease amino peptidase N, and mediatescell-viral membrane fusion. In particular, the spike protein contains anS1 subunit that facilitates binding of the coronavirus to cell surfaceproteins. Accordingly, the S1 subunit of the spike protein controlswhich cells are infected by the coronavirus. The spike protein alsocontains a S2 subunit, which is a transmembrane subunit that facilitatesviral and cellular membrane fusion. Accordingly, the MERS-CoV antigencan comprise a MERS-CoV spike protein, a S1 subunit of a MERS-CoV spikeprotein, or a S2 subunit of a MERS-CoV spike protein.

Upon binding cell surface proteins and membrane fusion, the coronavirusenters the cell and its singled-stranded RNA genome is released into thecytoplasm of the infected cell. The singled-stranded RNA genome is apositive strand and thus, can be translated into a RNA polymerase, whichproduces additional viral RNAs that are minus strands. Accordingly, theMERS-CoV antigen can also be a MERS-CoV RNA polymerase.

The viral minus RNA strands are transcribed into smaller, subgenomicpositive RNA strands, which are used to translate other viral proteins,for example, nucleocapsid (N) protein, envelope (E) protein, and matrix(M) protein. Accordingly, the MERS-CoV antigen can comprise a MERS-CoVnucleocapsid protein, a MERS-CoV envelope protein, or a MERS-CoV matrixprotein.

The viral minus RNA strands can also be used to replicate the viralgenome, which is bound by nucleocapsid protein. Matrix protein, alongwith spike protein, is integrated into the endoplasmic reticulum of theinfected cell. Together, the nucleocapsid protein bound to the viralgenome and the membrane-embedded matrix and spike proteins are buddedinto the lumen of the endoplasmic reticulum, thereby encasing the viralgenome in a membrane. The viral progeny are then transported by golgivesicles to the cell membrane of the infected cell and released into theextracellular space by endocytosis.

In some embodiments, the MERS-CoV antigen can be a MERS-CoV spikeprotein, a MERS-CoV RNA polymerase, a MERS-CoV nucleocapsid protein, aMERS-CoV envelope protein, a MERS-CoV matrix protein, a fragmentthereof, a variant thereof, or a combination thereof. The MERS-CoVantigen can be a consensus antigen derived from two or more MERS-CoVspike antigens, two or more MERS-CoV RNA polymerases, two or moreMERS-CoV nucleocapsid proteins, two or more envelope proteins, two ormore matrix proteins, or a combination thereof. The MERS-CoV consensusantigen can be modified for improved expression. Modification caninclude codon optimization, RNA optimization, addition of a kozaksequence for increased translation initiation, and/or the addition of animmunoglobulin leader sequence to increase the immunogenicity of theMERS-CoV antigen. In some embodiments the MERS-CoV antigen includes anIgE leader, which can be the amino acid sequence set forth in SEQ IDNO:6 and encoded by the nucleotide sequence set forth in SEQ ID NO:5.

(1) MERS-CoV Spike Antigen

The MERS-CoV antigen can be a MERS-CoV spike antigen, a fragmentthereof, a variant thereof, or a combination thereof. The MERS-CoV spikeantigen is capable of eliciting an immune response in a mammal againstone or more MERS-CoV strains. The MERS-CoV spike antigen can comprise anepitope(s) that makes it particularly effective as an immunogen againstwhich an anti-MERS-CoV immune response can be induced.

The MERS-CoV spike antigen can be a consensus sequence derived from twoor more strains of MERS-CoV. The MERS-CoV spike antigen can comprise aconsensus sequence and/or modification(s) for improved expression.Modification can include codon optimization, RNA optimization, additionof a kozak sequence for increased translation initiation, and/or theaddition of an immunoglobulin leader sequence to increase theimmunogenicity of the MERS-CoV spike antigen. The MERS-CoV consensusspike antigen can comprise a signal peptide such as an immunoglobulinsignal peptide, for example, but not limited to, an immunoglobulin E(IgE) or immunoglobulin (IgG) signal peptide. In some embodiments, theMERS-CoV consensus spike antigen can comprise a hemagglutinin (HA) tag.The MERS-CoV consensus spike antigen can be designed to elicit strongerand broader cellular and/or humoral immune responses than acorresponding codon optimized spike antigen.

The MERS-CoV consensus spike antigen can be the nucleic acid sequenceSEQ ID NO:1, which encodes SEQ ID NO:2 (FIGS. 8A and 8B). In someembodiments, the MERS-CoV consensus spike antigen can be the nucleicacid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity over an entire length of the nucleic acid sequence set forth inSEQ ID NO:1. In other embodiments, the MERS-CoV consensus spike antigencan be the nucleic acid sequence that encodes the amino acid sequencehaving at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity overan entire length of the amino acid sequence set forth in SEQ ID NO:2.

The MERS-CoV consensus spike antigen can be the amino acid sequence SEQID NO:2. In some embodiments, the MERS-CoV consensus spike antigen canbe the amino acid sequence having at least about 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identity over an entire length of the amino acidsequence set forth in SEQ ID NO:2.

Immunogenic fragments of SEQ ID NO:2 can be provided. Immunogenicfragments can comprise at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO:2. Insome embodiments, immunogenic fragments include a leader sequence, suchas for example an immunoglobulin leader, such as the IgE leader. In someembodiments, immunogenic fragments are free of a leader sequence.

Immunogenic fragments of proteins with amino acid sequences homologousto immunogenic fragments of SEQ ID NO:2 can be provided. Suchimmunogenic fragments can comprise at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% ofproteins that are 95% homologous to SEQ ID NO:2. Some embodiments relateto immunogenic fragments that have 96% homology to the immunogenicfragments of consensus protein sequences herein. Some embodiments relateto immunogenic fragments that have 97% homology to the immunogenicfragments of consensus protein sequences herein. Some embodiments relateto immunogenic fragments that have 98% homology to the immunogenicfragments of consensus protein sequences herein. Some embodiments relateto immunogenic fragments that have 99% homology to the immunogenicfragments of consensus protein sequences herein. In some embodiments,immunogenic fragments include a leader sequence, such as for example animmunoglobulin leader, such as the IgE leader. In some embodiments,immunogenic fragments are free of a leader sequence.

Some embodiments relate to immunogenic fragments of SEQ ID NO:1.Immunogenic fragments can be at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO:1.Immunogenic fragments can be at least 95%, at least 96%, at least 97% atleast 98% or at least 99% homologous to fragments of SEQ ID NO:1. Insome embodiments, immunogenic fragments include sequences that encode aleader sequence, such as for example an immunoglobulin leader, such asthe IgE leader. In some embodiments, fragments are free of codingsequences that encode a leader sequence.

(a) MERS-CoV Spike Antigen Lacking a Cytoplasmic Domain

The MERS-CoV antigen can be a MERS-CoV spike antigen lacking acytoplasmic domain (i.e., also referred to herein as “MERS-CoV spikeantigen ΔCD”), a fragment thereof, a variant thereof, or a combinationthereof. The MERS-CoV spike antigen ΔCD is capable of eliciting animmune response in a mammal against one or more MERS-CoV strains. TheMERS-CoV spike antigen ΔCD can comprise an epitope(s) that makes itparticularly effective as an immunogen against which an anti-MERS-CoVimmune response can be induced.

The MERS-CoV spike antigen ΔCD can be a consensus sequence derived fromtwo or more strains of MERS-CoV. The MERS-CoV spike antigen ΔCD cancomprise a consensus sequence and/or modification(s) for improvedexpression. Modification can include codon optimization, RNAoptimization, addition of a kozak sequence for increased translationinitiation, and/or the addition of an immunoglobulin leader sequence toincrease the immunogenicity of the MERS-CoV spike antigen ΔCD. TheMERS-CoV consensus spike antigen ΔCD can comprise a signal peptide suchas an immunoglobulin signal peptide, for example, but not limited to, animmunoglobulin E (IgE) or immunoglobulin (IgG) signal peptide. In someembodiments, the consensus spike antigen ΔCD can comprise ahemagglutinin (HA) tag. The MERS-CoV consensus spike antigen ΔCD can bedesigned to elicit stronger and broader cellular and/or humoral immuneresponses than a corresponding codon optimized spike antigen ΔCD.

The MERS-CoV consensus spike antigen ΔCD can be the nucleic acidsequence SEQ ID NO:3, which encodes SEQ ID NO:4 (FIGS. 8C and 8D). Insome embodiments, the MERS-CoV consensus spike antigen ΔCD can be thenucleic acid sequence having at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identity over an entire length of thenucleic acid sequence set forth in SEQ ID NO:3. In other embodiments,the MERS-CoV consensus spike antigen ΔCD can be the nucleic acidsequence that encodes the amino acid sequence having at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over anentire length of the amino acid sequence set forth in SEQ ID NO:4.

The MERS-CoV consensus spike antigen ΔCD can be the amino acid sequenceSEQ ID NO:4. In some embodiments, the MERS-CoV consensus spike antigenΔCD can be the amino acid sequence having at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entirelength of the amino acid sequence set forth in SEQ ID NO:4.

Immunogenic fragments of SEQ ID NO:4 can be provided. Immunogenicfragments can comprise at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO:4. Insome embodiments, immunogenic fragments include a leader sequence, suchas for example an immunoglobulin leader, such as the IgE leader. In someembodiments, immunogenic fragments are free of a leader sequence.

Immunogenic fragments of proteins with amino acid sequences homologousto immunogenic fragments of SEQ ID NO:4 can be provided. Suchimmunogenic fragments can comprise at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% ofproteins that are 95% homologous to SEQ ID NO:4. Some embodiments relateto immunogenic fragments that have 96% homology to the immunogenicfragments of consensus protein sequences herein. Some embodiments relateto immunogenic fragments that have 97% homology to the immunogenicfragments of consensus protein sequences herein. Some embodiments relateto immunogenic fragments that have 98% homology to the immunogenicfragments of consensus protein sequences herein. Some embodiments relateto immunogenic fragments that have 99% homology to the immunogenicfragments of consensus protein sequences herein. In some embodiments,immunogenic fragments include a leader sequence, such as for example animmunoglobulin leader, such as the IgE leader. In some embodiments,immunogenic fragments are free of a leader sequence.

Some embodiments relate to immunogenic fragments of SEQ ID NO:3.Immunogenic fragments can be at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO:3.Immunogenic fragments can be at least 95%, at least 96%, at least 97% atleast 98% or at least 99% homologous to fragments of SEQ ID NO:3. Insome embodiments, immunogenic fragments include sequences that encode aleader sequence, such as for example an immunoglobulin leader, such asthe IgE leader. In some embodiments, fragments are free of codingsequences that encode a leader sequence.

b. Vector

The vaccine can comprise one or more vectors that include a nucleic acidencoding the antigen. The one or more vectors can be capable ofexpressing the antigen. The vector can have a nucleic acid sequencecontaining an origin of replication. The vector can be a plasmid,bacteriophage, bacterial artificial chromosome or yeast artificialchromosome. The vector can be either a self-replicating extrachromosomalvector or a vector which integrates into a host genome.

The one or more vectors can be an expression construct, which isgenerally a plasmid that is used to introduce a specific gene into atarget cell. Once the expression vector is inside the cell, the proteinthat is encoded by the gene is produced by the cellular-transcriptionand translation machinery ribosomal complexes. The plasmid is frequentlyengineered to contain regulatory sequences that act as enhancer andpromoter regions and lead to efficient transcription of the gene carriedon the expression vector. The vectors of the present invention expresslarge amounts of stable messenger RNA, and therefore proteins.

The vectors may have expression signals such as a strong promoter, astrong termination codon, adjustment of the distance between thepromoter and the cloned gene, and the insertion of a transcriptiontermination sequence and a PTIS (portable translation initiationsequence).

(1) Expression Vectors

The vector can be a circular plasmid or a linear nucleic acid. Thecircular plasmid and linear nucleic acid are capable of directingexpression of a particular nucleotide sequence in an appropriate subjectcell. The vector can have a promoter operably linked to theantigen-encoding nucleotide sequence, which may be operably linked totermination signals. The vector can also contain sequences required forproper translation of the nucleotide sequence. The vector comprising thenucleotide sequence of interest may be chimeric, meaning that at leastone of its components is heterologous with respect to at least one ofits other components. The expression of the nucleotide sequence in theexpression cassette may be under the control of a constitutive promoteror of an inducible promoter, which initiates transcription only when thehost cell is exposed to some particular external stimulus. In the caseof a multicellular organism, the promoter can also be specific to aparticular tissue or organ or stage of development.

(2) Circular and Linear Vectors

The vector may be a circular plasmid, which may transform a target cellby integration into the cellular genome or exist extrachromosomally(e.g., autonomous replicating plasmid with an origin of replication).

The vector can be pVAX, pcDNA3.0, or provax, or any other expressionvector capable of expressing DNA encoding the antigen and enabling acell to translate the sequence to an antigen that is recognized by theimmune system.

Also provided herein is a linear nucleic acid vaccine, or linearexpression cassette (“LEC”), that is capable of being efficientlydelivered to a subject via electroporation and expressing one or moredesired antigens. The LEC may be any linear DNA devoid of any phosphatebackbone. The DNA may encode one or more antigens. The LEC may contain apromoter, an intron, a stop codon, and/or a polyadenylation signal. Theexpression of the antigen may be controlled by the promoter. The LEC maynot contain any antibiotic resistance genes and/or a phosphate backbone.The LEC may not contain other nucleic acid sequences unrelated to thedesired antigen gene expression.

The LEC may be derived from any plasmid capable of being linearized. Theplasmid may be capable of expressing the antigen. The plasmid can be pNP(Puerto Rico/34) or pM2 (New Caledonia/99). The plasmid may be WLV009,pVAX, pcDNA3.0, or provax, or any other expression vector capable ofexpressing DNA encoding the antigen and enabling a cell to translate thesequence to an antigen that is recognized by the immune system.

The LEC can be perM2. The LEC can be perNP. perNP and perMR can bederived from pNP (Puerto Rico/34) and pM2 (New Caledonia/99),respectively.

(3) Promoter, Intron, Stop Codon, and Polyadenylation Signal

The vector may have a promoter. A promoter may be any promoter that iscapable of driving gene expression and regulating expression of theisolated nucleic acid. Such a promoter is a cis-acting sequence elementrequired for transcription via a DNA dependent RNA polymerase, whichtranscribes the antigen sequence described herein. Selection of thepromoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter may be positionedabout the same distance from the transcription start in the vector as itis from the transcription start site in its natural setting. However,variation in this distance may be accommodated without loss of promoterfunction.

The promoter may be operably linked to the nucleic acid sequenceencoding the antigen and signals required for efficient polyadenylationof the transcript, ribosome binding sites, and translation termination.The promoter may be a CMV promoter, SV40 early promoter, SV40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or another promotershown effective for expression in eukaryotic cells.

The vector may include an enhancer and an intron with functional splicedonor and acceptor sites. The vector may contain a transcriptiontermination region downstream of the structural gene to provide forefficient termination. The termination region may be obtained from thesame gene as the promoter sequence or may be obtained from differentgenes.

c. Excipients and Other Components of the Vaccine

The vaccine may further comprise a pharmaceutically acceptableexcipient. The pharmaceutically acceptable excipient can be functionalmolecules such as vehicles, carriers, or diluents. The pharmaceuticallyacceptable excipient can be a transfection facilitating agent, which caninclude surface active agents, such as immune-stimulating complexes(ISCOMS), Freunds incomplete adjuvant, LPS analog includingmonophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles suchas squalene and squalene, hyaluronic acid, lipids, liposomes, calciumions, viral proteins, polyanions, polycations, or nanoparticles, orother known transfection facilitating agents.

The transfection facilitating agent is a polyanion, polycation,including poly-L-glutamate (LGS), or lipid. The transfectionfacilitating agent is poly-L-glutamate, and the poly-L-glutamate may bepresent in the vaccine at a concentration less than 6 mg/ml. Thetransfection facilitating agent may also include surface active agentssuch as immune-stimulating complexes (ISCOMS), Freunds incompleteadjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides,quinone analogs and vesicles such as squalene and squalene, andhyaluronic acid may also be used administered in conjunction with thegenetic construct. The DNA plasmid vaccines may also include atransfection facilitating agent such as lipids, liposomes, includinglecithin liposomes or other liposomes known in the art, as aDNA-liposome mixture (see for example WO9324640), calcium ions, viralproteins, polyanions, polycations, or nanoparticles, or other knowntransfection facilitating agents. The transfection facilitating agent isa polyanion, polycation, including poly-L-glutamate (LGS), or lipid.Concentration of the transfection agent in the vaccine is less than 4mg/ml, less than 2 mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, lessthan 0.500 mg/ml, less than 0.250 mg/ml, less than 0.100 mg/ml, lessthan 0.050 mg/ml, or less than 0.010 mg/ml.

The pharmaceutically acceptable excipient can be an adjuvant. Theadjuvant can be other genes that are expressed in an alternative plasmidor are delivered as proteins in combination with the plasmid above inthe vaccine. The adjuvant may be selected from the group consisting of:α-interferon(IFN-α), β-interferon (IFN-β), γ-interferon, plateletderived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growthfactor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelialthymus-expressed chemokine (TECK), mucosae-associated epithelialchemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL-15 havingthe signal sequence deleted and optionally including the signal peptidefrom IgE. The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, plateletderived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growthfactor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or acombination thereof.

Other genes that can be useful as adjuvants include those encoding:MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin,CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1,ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18,CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7,IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNFreceptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF,DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1,Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K,SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec,TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND,NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 andfunctional fragments thereof.

The vaccine may further comprise a genetic vaccine facilitator agent asdescribed in U.S. Ser. No. 021,579 filed Apr. 1, 1994, which is fullyincorporated by reference.

The vaccine can be formulated according to the mode of administration tobe used. An injectable vaccine pharmaceutical composition can besterile, pyrogen free and particulate free. An isotonic formulation orsolution can be used. Additives for isotonicity can include sodiumchloride, dextrose, mannitol, sorbitol, and lactose. The vaccine cancomprise a vasoconstriction agent. The isotonic solutions can includephosphate buffered saline. Vaccine can further comprise stabilizersincluding gelatin and albumin. The stabilizers can allow the formulationto be stable at room or ambient temperature for extended periods oftime, including LGS or polycations or polyanions.

3. METHOD OF VACCINATION

Also provided herein is a method of treating, protecting against, and/orpreventing disease in a subject in need thereof by administering thevaccine to the subject. Administration of the vaccine to the subject caninduce or elicit an immune response in the subject. The induced immuneresponse can be used to treat, prevent, and/or protect against disease,for example, pathologies relating to MERS-CoV infection. The inducedimmune response provided the subject administered the vaccine resistanceto one or more MERS-CoV strains.

The induced immune response can include an induced humoral immuneresponse and/or an induced cellular immune response. The humoral immuneresponse can be induced by about 1.5-fold to about 16-fold, about 2-foldto about 12-fold, or about 3-fold to about 10-fold. The induced humoralimmune response can include IgG antibodies and/or neutralizingantibodies that are reactive to the antigen. The induced cellular immuneresponse can include a CD8⁺ T cell response, which is induced by about2-fold to about 30-fold, about 3-fold to about 25-fold, or about 4-foldto about 20-fold.

The vaccine dose can be between 1 μg to 10 mg active component/kg bodyweight/time, and can be 20 μg to 10 mg component/kg body weight/time.The vaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, or 31 days. The number of vaccine doses for effective treatment canbe 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

a. Administration

The vaccine can be formulated in accordance with standard techniqueswell known to those skilled in the pharmaceutical art. Such compositionscan be administered in dosages and by techniques well known to thoseskilled in the medical arts taking into consideration such factors asthe age, sex, weight, and condition of the particular subject, and theroute of administration. The subject can be a mammal, such as a human, ahorse, a cow, a pig, a sheep, a cat, a dog, a rat, or a mouse.

The vaccine can be administered prophylactically or therapeutically. Inprophylactic administration, the vaccines can be administered in anamount sufficient to induce an immune response. In therapeuticapplications, the vaccines are administered to a subject in need thereofin an amount sufficient to elicit a therapeutic effect. An amountadequate to accomplish this is defined as “therapeutically effectivedose.” Amounts effective for this use will depend on, e.g., theparticular composition of the vaccine regimen administered, the mannerof administration, the stage and severity of the disease, the generalstate of health of the patient, and the judgment of the prescribingphysician.

The vaccine can be administered by methods well known in the art asdescribed in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997));Felgner et al. (U.S. Pat. No. 5,580,859, issued Dec. 3, 1996); Felgner(U.S. Pat. No. 5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S.Pat. No. 5,679,647, issued Oct. 21, 1997), the contents of all of whichare incorporated herein by reference in their entirety. The DNA of thevaccine can be complexed to particles or beads that can be administeredto an individual, for example, using a vaccine gun. One skilled in theart would know that the choice of a pharmaceutically acceptable carrier,including a physiologically acceptable compound, depends, for example,on the route of administration of the expression vector.

The vaccine can be delivered via a variety of routes. Typical deliveryroutes include parenteral administration, e.g., intradermal,intramuscular or subcutaneous delivery. Other routes include oraladministration, intranasal, and intravaginal routes. For the DNA of thevaccine in particular, the vaccine can be delivered to the interstitialspaces of tissues of an individual (Felgner et al., U.S. Pat. Nos.5,580,859 and 5,703,055, the contents of all of which are incorporatedherein by reference in their entirety). The vaccine can also beadministered to muscle, or can be administered via intradermal orsubcutaneous injections, or transdermally, such as by iontophoresis.Epidermal administration of the vaccine can also be employed. Epidermaladministration can involve mechanically or chemically irritating theoutermost layer of epidermis to stimulate an immune response to theirritant (Carson et al., U.S. Pat. No. 5,679,647, the contents of whichare incorporated herein by reference in its entirety).

The vaccine can also be formulated for administration via the nasalpassages. Formulations suitable for nasal administration, wherein thecarrier is a solid, can include a coarse powder having a particle size,for example, in the range of about 10 to about 500 microns which isadministered in the manner in which snuff is taken, i.e., by rapidinhalation through the nasal passage from a container of the powder heldclose up to the nose. The formulation can be a nasal spray, nasal drops,or by aerosol administration by nebulizer. The formulation can includeaqueous or oily solutions of the vaccine.

The vaccine can be a liquid preparation such as a suspension, syrup orelixir. The vaccine can also be a preparation for parenteral,subcutaneous, intradermal, intramuscular or intravenous administration(e.g., injectable administration), such as a sterile suspension oremulsion.

The vaccine can be incorporated into liposomes, microspheres or otherpolymer matrices (Felgner et al., U.S. Pat. No. 5,703,055; Gregoriadis,Liposome Technology, Vols. I to III (2nd ed. 1993), the contents ofwhich are incorporated herein by reference in their entirety). Liposomescan consist of phospholipids or other lipids, and can be nontoxic,physiologically acceptable and metabolizable carriers that arerelatively simple to make and administer.

The vaccine can be administered via electroporation, such as by a methoddescribed in U.S. Pat. No. 7,664,545, the contents of which areincorporated herein by reference. The electroporation can be by a methodand/or apparatus described in U.S. Pat. Nos. 6,302,874; 5,676,646;6,241,701; 6,233,482; 6,216,034; 6,208,893; 6,192,270; 6,181,964;6,150,148; 6,120,493; 6,096,020; 6,068,650; and 5,702,359, the contentsof which are incorporated herein by reference in their entirety. Theelectroporation may be carried out via a minimally invasive device.

The minimally invasive electroporation device (“MID”) may be anapparatus for injecting the vaccine described above and associated fluidinto body tissue. The device may comprise a hollow needle, DNA cassette,and fluid delivery means, wherein the device is adapted to actuate thefluid delivery means in use so as to concurrently (for example,automatically) inject DNA into body tissue during insertion of theneedle into the said body tissue. This has the advantage that theability to inject the DNA and associated fluid gradually while theneedle is being inserted leads to a more even distribution of the fluidthrough the body tissue. The pain experienced during injection may bereduced due to the distribution of the DNA being injected over a largerarea.

The MID may inject the vaccine into tissue without the use of a needle.The MID may inject the vaccine as a small stream or jet with such forcethat the vaccine pierces the surface of the tissue and enters theunderlying tissue and/or muscle. The force behind the small stream orjet may be provided by expansion of a compressed gas, such as carbondioxide through a micro-orifice within a fraction of a second. Examplesof minimally invasive electroporation devices, and methods of usingthem, are described in published U.S. Patent Application No.20080234655; U.S. Pat. No. 6,520,950; U.S. Pat. No. 7,171,264; U.S. Pat.No. 6,208,893; U.S. Pat. No. 6,009,347; U.S. Pat. No. 6,120,493; U.S.Pat. No. 7,245,963; U.S. Pat. No. 7,328,064; and U.S. Pat. No.6,763,264, the contents of each of which are herein incorporated byreference.

The MID may comprise an injector that creates a high-speed jet of liquidthat painlessly pierces the tissue. Such needle-free injectors arecommercially available. Examples of needle-free injectors that can beutilized herein include those described in U.S. Pat. Nos. 3,805,783;4,447,223; 5,505,697; and 4,342,310, the contents of each of which areherein incorporated by reference.

A desired vaccine in a form suitable for direct or indirectelectrotransport may be introduced (e.g., injected) using a needle-freeinjector into the tissue to be treated, usually by contacting the tissuesurface with the injector so as to actuate delivery of a jet of theagent, with sufficient force to cause penetration of the vaccine intothe tissue. For example, if the tissue to be treated is mucosa, skin ormuscle, the agent is projected towards the mucosal or skin surface withsufficient force to cause the agent to penetrate through the stratumcorneum and into dermal layers, or into underlying tissue and muscle,respectively.

Needle-free injectors are well suited to deliver vaccines to all typesof tissues, particularly to skin and mucosa. In some embodiments, aneedle-free injector may be used to propel a liquid that contains thevaccine to the surface and into the subject's skin or mucosa.Representative examples of the various types of tissues that can betreated using the invention methods include pancreas, larynx,nasopharynx, hypopharynx, oropharynx, lip, throat, lung, heart, kidney,muscle, breast, colon, prostate, thymus, testis, skin, mucosal tissue,ovary, blood vessels, or any combination thereof.

The MID may have needle electrodes that electroporate the tissue. Bypulsing between multiple pairs of electrodes in a multiple electrodearray, for example set up in rectangular or square patterns, providesimproved results over that of pulsing between a pair of electrodes.Disclosed, for example, in U.S. Pat. No. 5,702,359 entitled “NeedleElectrodes for Mediated Delivery of Drugs and Genes” is an array ofneedles wherein a plurality of pairs of needles may be pulsed during thetherapeutic treatment. In that application, which is incorporated hereinby reference as though fully set forth, needles were disposed in acircular array, but have connectors and switching apparatus enabling apulsing between opposing pairs of needle electrodes. A pair of needleelectrodes for delivering recombinant expression vectors to cells may beused. Such a device and system is described in U.S. Pat. No. 6,763,264,the contents of which are herein incorporated by reference.Alternatively, a single needle device may be used that allows injectionof the DNA and electroporation with a single needle resembling a normalinjection needle and applies pulses of lower voltage than thosedelivered by presently used devices, thus reducing the electricalsensation experienced by the patient.

The MID may comprise one or more electrode arrays. The arrays maycomprise two or more needles of the same diameter or differentdiameters. The needles may be evenly or unevenly spaced apart. Theneedles may be between 0.005 inches and 0.03 inches, between 0.01 inchesand 0.025 inches; or between 0.015 inches and 0.020 inches. The needlemay be 0.0175 inches in diameter. The needles may be 0.5 mm, 1.0 mm, 1.5mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more spaced apart.

The MID may consist of a pulse generator and a two or more-needlevaccine injectors that deliver the vaccine and electroporation pulses ina single step. The pulse generator may allow for flexible programming ofpulse and injection parameters via a flash card operated personalcomputer, as well as comprehensive recording and storage ofelectroporation and patient data. The pulse generator may deliver avariety of volt pulses during short periods of time. For example, thepulse generator may deliver three 15 volt pulses of 100 ms in duration.An example of such a MID is the Elgen 1000 system by Inovio BiomedicalCorporation, which is described in U.S. Pat. No. 7,328,064, the contentsof which are herein incorporated by reference.

The MID may be a CELLECTRA (Inovio Pharmaceuticals, Blue Bell Pa.)device and system, which is a modular electrode system, that facilitatesthe introduction of a macromolecule, such as a DNA, into cells of aselected tissue in a body or plant. The modular electrode system maycomprise a plurality of needle electrodes; a hypodermic needle; anelectrical connector that provides a conductive link from a programmableconstant-current pulse controller to the plurality of needle electrodes;and a power source. An operator can grasp the plurality of needleelectrodes that are mounted on a support structure and firmly insertthem into the selected tissue in a body or plant. The macromolecules arethen delivered via the hypodermic needle into the selected tissue. Theprogrammable constant-current pulse controller is activated andconstant-current electrical pulse is applied to the plurality of needleelectrodes. The applied constant-current electrical pulse facilitatesthe introduction of the macromolecule into the cell between theplurality of electrodes. Cell death due to overheating of cells isminimized by limiting the power dissipation in the tissue by virtue ofconstant-current pulses. The Cellectra device and system is described inU.S. Pat. No. 7,245,963, the contents of which are herein incorporatedby reference.

The MID may be an Elgen 1000 system (Inovio Pharmaceuticals). The Elgen1000 system may comprise device that provides a hollow needle; and fluiddelivery means, wherein the apparatus is adapted to actuate the fluiddelivery means in use so as to concurrently (for example automatically)inject fluid, the described vaccine herein, into body tissue duringinsertion of the needle into the said body tissue. The advantage is theability to inject the fluid gradually while the needle is being insertedleads to a more even distribution of the fluid through the body tissue.It is also believed that the pain experienced during injection isreduced due to the distribution of the volume of fluid being injectedover a larger area.

In addition, the automatic injection of fluid facilitates automaticmonitoring and registration of an actual dose of fluid injected. Thisdata can be stored by a control unit for documentation purposes ifdesired.

It will be appreciated that the rate of injection could be either linearor non-linear and that the injection may be carried out after theneedles have been inserted through the skin of the subject to be treatedand while they are inserted further into the body tissue.

Suitable tissues into which fluid may be injected by the apparatus ofthe present invention include tumor tissue, skin or liver tissue but maybe muscle tissue.

The apparatus further comprises needle insertion means for guidinginsertion of the needle into the body tissue. The rate of fluidinjection is controlled by the rate of needle insertion. This has theadvantage that both the needle insertion and injection of fluid can becontrolled such that the rate of insertion can be matched to the rate ofinjection as desired. It also makes the apparatus easier for a user tooperate. If desired means for automatically inserting the needle intobody tissue could be provided.

A user could choose when to commence injection of fluid. Ideallyhowever, injection is commenced when the tip of the needle has reachedmuscle tissue and the apparatus may include means for sensing when theneedle has been inserted to a sufficient depth for injection of thefluid to commence. This means that injection of fluid can be prompted tocommence automatically when the needle has reached a desired depth(which will normally be the depth at which muscle tissue begins). Thedepth at which muscle tissue begins could for example be taken to be apreset needle insertion depth such as a value of 4 mm which would bedeemed sufficient for the needle to get through the skin layer.

The sensing means may comprise an ultrasound probe. The sensing meansmay comprise a means for sensing a change in impedance or resistance. Inthis case, the means may not as such record the depth of the needle inthe body tissue but will rather be adapted to sense a change inimpedance or resistance as the needle moves from a different type ofbody tissue into muscle. Either of these alternatives provides arelatively accurate and simple to operate means of sensing thatinjection may commence. The depth of insertion of the needle can furtherbe recorded if desired and could be used to control injection of fluidsuch that the volume of fluid to be injected is determined as the depthof needle insertion is being recorded.

The apparatus may further comprise: a base for supporting the needle;and a housing for receiving the base therein, wherein the base ismoveable relative to the housing such that the needle is retractedwithin the housing when the base is in a first rearward positionrelative to the housing and the needle extends out of the housing whenthe base is in a second forward position within the housing. This isadvantageous for a user as the housing can be lined up on the skin of apatient, and the needles can then be inserted into the patient's skin bymoving the housing relative to the base.

As stated above, it is desirable to achieve a controlled rate of fluidinjection such that the fluid is evenly distributed over the length ofthe needle as it is inserted into the skin. The fluid delivery means maycomprise piston driving means adapted to inject fluid at a controlledrate. The piston driving means could for example be activated by a servomotor. However, the piston driving means may be actuated by the basebeing moved in the axial direction relative to the housing. It will beappreciated that alternative means for fluid delivery could be provided.Thus, for example, a closed container which can be squeezed for fluiddelivery at a controlled or non-controlled rate could be provided in theplace of a syringe and piston system.

The apparatus described above could be used for any type of injection.It is however envisaged to be particularly useful in the field ofelectroporation and so it may further comprises means for applying avoltage to the needle. This allows the needle to be used not only forinjection but also as an electrode during, electroporation. This isparticularly advantageous as it means that the electric field is appliedto the same area as the injected fluid. There has traditionally been aproblem with electroporation in that it is very difficult to accuratelyalign an electrode with previously injected fluid and so users havetended to inject a larger volume of fluid than is required over a largerarea and to apply an electric field over a higher area to attempt toguarantee an overlap between the injected substance and the electricfield. Using the present invention, both the volume of fluid injectedand the size of electric field applied may be reduced while achieving agood fit between the electric field and the fluid.

4. KIT

Provided herein is a kit, which can be used for treating a subject usingthe method of vaccination described above. The kit can comprise thevaccine.

The kit can also comprise instructions for carrying out the vaccinationmethod described above and/or how to use the kit. Instructions includedin the kit can be affixed to packaging material or can be included as apackage insert. While instructions are typically written or printedmaterials, they are not limited to such. Any medium capable of storinginstructions and communicating them to an end user is contemplated bythis disclosure. Such media include, but are not limited to, electronicstorage media (e.g., magnetic discs, tapes, cartridges), optical media(e.g., CD ROM), and the like. As used herein, the term “instructions”can include the address of an internet site which provides instructions.

The present invention has multiple aspects, illustrated by the followingnon-limiting examples.

5. EXAMPLES Example 1 MERS-CoV Consensus Spike Antigen

As described elsewhere herein, multiple strains of MERS-CoV exist asshown in FIG. 3 and a consensus spike antigen was created to account forvariance between these multiple strains of MERS-CoV. Accordingly, theMERS-CoV consensus spike antigen was derived from the respective spikeantigens of the viruses listed in FIG. 3. Phylogenetic analysis wasperformed based upon the spike antigen and placed the MERS-CoV consensusspike antigen relative to its component spike antigens (FIG. 3, label“MERS-HCoV-Consensus” represented the MERS-CoV consensus spike antigen).The MERS-CoV consensus spike antigen also contained an N-terminalimmunoglobulin E (IgE) leader sequence. The nucleic acid and amino acidsequences comprising the MERS-CoV consensus spike antigen are shown inFIGS. 8A and 8B, respectively. In FIG. 8A, underlining indicates thenucleotides that encode the IgE leader sequence. In FIG. 8B, underliningindicates the IgE leader sequence.

A kozak sequence was placed on the 5′ end of the nucleic acid sequenceencoding the MERS-CoV consensus spike antigen and the resulting nucleicacid sequence was inserted between the BamHI and XhoI sites of the pVAX1vector (Life Technologies, Carlsbad, Calif.) (FIG. 4A, resultingconstruct named MERS-HCoV-WT). Correct insertion of this nucleic acidsequence into the pVAX1 vector was confirmed by BamHI and XhoIdigestion, followed by gel electrophoresis (FIG. 4B). In FIG. 4B, Lane Mindicated the marker, lane 1 was undigested MERS-HCoV-WT construct, andlane 2 was digested MERS-HCoV-WT construct. Digestion yielded theexpected fragment sizes, indicating that the MERS-HCoV-WT constructincluded the pVAX1 vector and the inserted nucleic acid sequence (i.e.,the nucleic acid sequence containing the kozak sequence and encoding theMERS-CoV consensus spike antigen).

Construction and characterization of this MERS-HCoV-WT construct is alsodescribed below in Examples 9 and 10.

Example 2 MERS-CoV Consensus Spike Antigen Lacking a Cytoplasmic Domain

The spike antigen from coronaviruses is a type 1 membrane glycoproteinthat has a transmembrane domain, which in turn, defines cytoplasmic andnon-cytoplasmic domains of the spike antigen. To determine the locationof the cytoplasmic domain in the MERS-CoV spike antigen, web-basedsoftware was used to predict the location of domains within variousMERS-CoV spike antigens. In particular, the web-based software Phobiusand InterProScan were employed in this analysis. Representative resultsfrom the Phobius and InterProScan analysis are shown in FIGS. 5 and 6,respectively.

From this domain analysis of the MERS-CoV spike antigens, a secondMERS-CoV consensus spike antigen was generated, which lacked thecytoplasmic domain. In particular, the nucleic acid sequence encodingthe MERS-CoV consensus spike antigen (described in Example 1, FIG. 8A,SEQ ID NO:1) was modified to insert two stop codons such that thetranslated protein did not contain the cytoplasmic domain (i.e.,MERS-CoV consensus spike antigen ΔCD).

The nucleic acid and amino acid sequences of the MERS-CoV consensusspike antigen ΔCD are shown in FIGS. 8C and 8D, respectively. In FIG.8C, underlining indicates the nucleotides that encode the IgE leadersequence and double underlining indicates the two inserted stop codons(relative to SEQ ID NO:1 that prevented translation of the cytoplasmicdomain). In FIG. 8D, underlining indicates the IgE leader sequence. Aschematic illustrating the resulting construct, MERS-HCoV-ΔCD, is shownin FIG. 7A.

The MERS-HCoV-ΔCD construct was digested with BamHI and XhoI, followedby gel electrophoresis, to confirm the insert was present in the pVAX1vector (FIG. 7B). In FIG. 7B, Lane M indicated the marker, lane 1 wasundigested MERS-HCoV-ΔCD construct, and lane 2 was digestedMERS-HCoV-ΔCD construct. Digestion yielded the expected fragment sizes,indicating that the MERS-HCoV-ΔCD construct included the pVAX1 vectorand the inserted nucleic acid sequence (i.e., the nucleic acid sequencecontaining the kozak sequence and encoding the MERS-CoV consensus spikeantigen ΔCD).

Construction and characterization of this MERS-HCoV-ΔCD construct isalso described below in Examples 9 and 10.

Example 3 Expression and Humoral Response

The above described MERS-HCoV-WT and MERS-HCoV-ΔCD constructs wereexamined to determine that the respective antigens were expressed withincells and recognizable by antibody. The MERS-HCoV-WT and MERS-HCoV-ΔCDconstructs, along with pVAX1, were transfected into 293T cells. pVAX1served as a control.

Cell lysates were prepared two days post transfection and run on a 5-15%SDS gel. In particular, 10 μg, 25 μg, and 50 μg of cell lysate wereloaded into separate wells for the cell lysates obtained from the 293Tcells transfected with either the MERS-HCoV-WT or MERS-HCoV-ΔCDconstruct. Immunoblot analysis was then performed using sera from miceimmunized with the MERS-HCoV-WT construct. No protein bands weredetected in the cell lysates obtained from 293T cells transfected withpVAX1 (FIG. 9, lane labeled pVAX1). For both the MERS-HCoV-WT andMERS-HCoV-ΔCD constructs, protein bands were detected that correspondedto the predicted molecular weights of the respective antigens, therebyconfirming expression of the respective antigens from the MERS-HCoV-WTand MERS-HCoV-ΔCD constructs (FIG. 9).

The immunoblot also indicated that the sera from the mice immunized withthe MERS-HCoV-WT construct recognized both the MERS-CoV consensus spikeantigen and the MERS-CoV consensus spike antigen ΔCD, but not otherproteins in the cell lysates (as evidenced by no observed bands in thecell lysate obtained from 293T cells transfected with pVAX1).Accordingly, this sera was specific to the consensus spike antigens. Theimmunoblot further indicated that the MERS-HCoV-WT construct wasimmunogenic and produced a strong humoral response.

Example 4 Immunization Schedule for Examples 5-7

C57/BL6 mice were immunized with pVAX1, MERS-HCoV-WT construct, orMERS-HCoV-ΔCD following the immunization regimen illustrated in FIG. 10.On day 0, each mouse was given its respective vaccine intramuscularly(IM), followed by electroporation. In particular, each mouse wasanesthetized with tribromoethanol-avertin and the hair located in thearea of the tibialis anterior (TA) muscle was shaved using a smallanimal clipper to expose the skin. The vaccine was administered in afinal volume of 30 to 50 μL via IM injection. A sterile CELLECTRA 3P IDarray was then inserted through the skin into the muscle surrounding theIM injection site. The CELLECTRA 3P ID array included three 26 gaugeneedle-electrodes that were 3 mm in length and held together by moldedplastic. The needle-electrodes were attached to the CELLECTRAelectroporation device with the CELLECTRA 3P application. Afterinsertion of the array into the muscle, a brief electric pulse wasdelivered and the immunized mice were placed into their respective cagesand carefully observed until consciousness was regained. The aboveimmunization procedure was repeated on day 14 and day 28. Accordingly,the immunization on day 0 was a priming immunization and theimmunizations on days 14 and 28 were boost immunizations. At day 35, themice were sacrificed and immune analysis as described below in Examples5-7 was performed.

Example 5 IgG Antibody Response

To further examine the humoral immune response induced by theMERS-HCoV-WT and MERS-HCoV-ΔCD constructs, mice were immunized asdescribed above in Example 4. A pre-bleed was obtained before initiationof the immunization regimen and bleeds were also obtained on day 21 andday 35 of the immunization regimen. The pre-bleed and bleeds wereobtained via a retro orbital method.

The pre-bleed, day 21 bleed, and day 35 bleed from the mice immunizedwith the MERS-HCoV-WT construct were examined to determine the level ofIgG antibodies that were immunoreactive with peptides derived from thefull-length consensus spike antigen. Specifically, sera from thepre-bleed and each bleed were diluted 1:50 and added to ELISA platesthat were coated with mixed peptide pools. The mixed peptide poolscontained peptides derived from different portions of the full-lengthconsensus spike antigen. Specifically, the mixed peptide pools were anequal mixture of the 6 linear peptide pools 1-6 described below inExample 6 and FIG. 15.

As shown in FIG. 11A, the MERS-HCoV-WT construct elicited high levels ofIgG antibody that were immunoreactive with peptides derived from thefull-length consensus spike antigen. The data in FIG. 11A are from thesera of 4 mice and represented the mean±standard error of the mean(SEM). These data indicated (via comparison of the pre-bleed and bleeds)that the MERS-HCoV-WT construct induced an about 4-fold to about 6-foldincrease in the level of IgG antibody that recognized the peptidesderived from the full-length consensus spike antigen.

The pre-bleed, day 21 bleed, and day 35 bleed from mice immunized withthe MERS-HCoV-ΔCD construct were also examined to determine the level ofIgG antibodies that were immunoreactive with peptides derived from thefull-length consensus spike antigen. Sera from the pre-bleed and eachbleed were diluted 1:50 and added to ELISA plates that were coated withmixed peptide pools. The mixed peptide pools contained peptides derivedfrom different portions of the full-length consensus spike antigen.Specifically, the mixed peptide pools were an equal mixture of the 6linear peptide pools 1-6 described below in Example 6 and FIG. 15.

As shown in FIG. 11B, the MERS-HCoV-ΔCD construct elicited high levelsof IgG antibody that were immunoreactive with the peptides derived fromthe full-length consensus spike antigen. The data in FIG. 11B are fromthe sera of 4 mice and represented mean±SEM. These data indicated (viacomparison of the pre-bleed and bleeds) that the MERS-HCoV-ΔCD constructinduced an about 6-fold to about 8-fold increase in the level of IgGantibody that recognized the peptides derived from the full-lengthconsensus spike antigen.

In summary, the data in FIGS. 11A and 11B indicated that both theMERS-HCoV-WT and MERS-HCoV-ΔCD constructs elicited high levels of IgGantibodies that were immunoreactive with the peptides derived from thefull-length consensus spike antigen. These data also indicated that theMERS-HCoV-ΔCD construct elicited higher levels of immunoreactive IgGantibody than the MERS-HCoV-WT construct. Accordingly, the MERS-HCoV-ΔCDconstruct was more immunogenic than the MERS-HCoV-WT construct asmeasured by IgG antibody response.

Example 6 CD8⁺ T Cell Response

As described above, the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs eachinduced a significant humoral immune response directed towards theMERS-CoV spike antigen. To determine if the MERS-HCoV-WT andMERS-CoV-ΔCD constructs also induced a cellular immune response, micewere immunized as described above in Example 4. After sacrifice on day35 of the immunization regimen, the antigen-specific response of CD8⁺ Tcells was analyzed using an interferon-gamma (IFN-γ) ELISpot assay witha matrix of peptide pools covering the amino acid sequence of thefull-length consensus MERS-CoV spike antigen. The matrix of peptidepools is shown in FIG. 12 and also facilitated identification of thedominant epitope(s) recognized by the CD8⁺ T cell response that wasinduced by the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs. This matrix ofpeptide pools was derived from a peptide scan over the entire length ofthe MERS-HCoV-WT spike antigen and the peptides were 15-mers with 11amino acid overlap.

The results of the IFN-γ ELISpot assay with this matrix of peptide poolsare shown in FIGS. 13 and 14 for the MERS-HCoV-WT and MERS-HCoV-ΔCDconstructs, respectively. In both FIGS. 13 and 14, the data representedmean±SEM. Both constructs induced a significant CD8⁺ T cell response, inwhich the dominant epitope resided in pools 18, 19, 20, and 21 (FIGS. 13and 14, circled bars). Another epitope resided in pool 4. Accordingly,these data indicated that the MERS-HCoV-WT and MERS-HCoV-ΔCD constructsinduced a strong cellular immune response that was reactive to a subsetof spike antigen peptides.

To further examine the CD8⁺ T cell response, mice were immunized with 25μg of pVAX1, MERS-HCoV-WT construct, or MERS-HCoV-ΔCD constructfollowing the immunization regimen described in Example 4 above.Immunization with pVAX1 served as a control. After sacrifice on day 35of the immunization regimen, the antigen-specific response of CD8⁺ Tcells isolated from three groups of mice was examined using the IFN-γELISpot assay. In particular, the CD8⁺ T cells were stimulated with 6different peptide pools. These peptide pools 1-6 covered the peptidesshown in the matrix of FIG. 12.

As shown in FIG. 15, the magnitude of the cellular immune responseinduced by the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs varieddepending on the peptide pool used to stimulate the CD8⁺ T cellsisolated from the respective mice. For both the MERS-HCoV-WT andMERS-HCoV-ΔCD constructs, peptide pool 1 elicited a CD8⁺ T cell responsesimilar to the control pVAX1, thereby indicating that peptide pool 1 didnot contain an epitope that stimulated the CD8⁺ T cells. Peptide pools1-6 elicited similar responses from the CD8⁺ T cells isolated from miceimmunized with pVAX1.

Peptide pools 2, 3, and 4 elicited comparable responses from the CD8⁺ Tcells isolated from mice vaccinated with MERS-HCoV-WT or MERS-HCoV-ΔCDconstruct. Additionally, the CD8⁺ T cell response to peptide pools 2, 3,and 4 was significantly higher when the mice were immunized with eitherthe MERS-HCoV-WT or MERS-HCoV-ΔCD construct as compared to the pVAX1control (FIG. 15). In particular, the CD8⁺ T cell response to peptidepools 2, 3, and 4 was about 7-fold higher when the mice were immunizedwith the MERS-HCoV-ΔCD construct as compared to the control pVAX1. TheCD8⁺ T cell response to peptide pools 2, 3, and 4 was about 7.5-fold,about 9-fold, and about 10-fold higher, respectively, when the mice wereimmunized with the MERS-HCoV-WT construct as compared to the controlpVAX1. Accordingly, these data indicated that the MERS-HCoV-WT andMERS-HCoV-ΔCD constructs, unlike the control pVAX1, induced asignificant CD8⁺ T cell response to peptides contained within peptidepools 2, 3, and 4.

Peptide pools 5 and 6 elicited a greater response from the CD8⁺ T cellsisolated from mice immunized with the MERS-HCoV-WT or MERS-HCoV-ΔCDconstruct as compared to peptide pools 1, 2, 3, and 4 (FIG. 15).Additionally, the CD8⁺ T cell response to peptide pools 5 and 6 wassignificantly higher when the mice were immunized with either theMERS-HCoV-WT or MERS-HCoV-ΔCD construct as compared to the controlpVAX1. In particular, the CD8⁺ T cell response to peptide pools 5 and 6was about 9-fold and about 11-fold higher, respectively, when the micewere immunized with the MERS-HCoV-ΔCD construct as compared to thecontrol pVAX1. The CD8⁺ T cell response to peptide pools 5 and 6 wasabout 13-fold and about 15-fold higher, respectively, when the mice wereimmunized with the MERS-HCoV-WT construct as compared to the controlpVAX1. Accordingly, these data indicated that the MERS-HCoV-WT andMERS-HCoV-ΔCD constructs, unlike the control pVAX1, induced asignificant CD8⁺ T cell response to peptides contained within peptidepools 5 and 6.

In summary, the above data indicated that immunization with theMERS-HCoV-WT and MERS-HCoV-ΔCD constructs induced a significant cellularimmune response that was specific and reactive to a subset of spikeantigen peptides.

Example 7 Polyfunctional Cellular Immune Response

As described above, the MERS-HCoV-WT and MERS-HCoV-ΔCD constructsinduced a CD8⁺ T cell response that was reactive and specific to theMERS-CoV spike antigen. To further examine the cellular immune responseinduced by the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs, thefunctionality of the T cells after immunization was examined. Inparticular, mice were immunized with pVAX1, MERS-HCoV-WT construct, orMERS-HCoV-ΔCD construct following the immunization regimen describedabove in Example 4. At day 35 of the immunization regimen, splenocyteswere isolated from the sacrificed mice. The isolated splenocytes werestimulated in vitro with a peptide pool containing peptides derived fromthe full-length consensus MERS-CoV spike antigen. The peptide pool andthe splenocytes were incubated together for 5 hours. Cells were thenstained for intracellular production of IFN-γ, tumor necrosis factoralpha (TNF-α), and interleukin-2 (IL-2) and sorted byfluoresecence-activated cell sorting (FACS).

FIGS. 16A, 16B, 16C, and 16D show the measured frequency of CD3⁺CD4⁺ Tcells producing IFN-γ, TNF-α, IL-2, or both IFN-γ and TNF-α,respectively, in the stimulated splenocyte populations. The data inFIGS. 16A-16D represented, for each group, splenocytes isolated from 3mice. The data in FIGS. 16A-16D also represented mean±SEM. The frequencyof CD3⁺CD4⁺ T cells producing IFN-γ, TNF-α, IL-2, or both IFN-γ andTNF-α was significantly increased in the MERS-HCoV-WT and MERS-HCoV-ΔCDgroups as compared to the control pVAX1 group.

In particular, the frequency of CD3⁺CD4⁺IFN-γ⁺ T cells was about 6-foldand about 13-fold greater in the splenoctye populations isolated frommice immunized with the MERS-HCoV-ΔCD and MERS-HCoV-WT constructs,respectively, as compared to the control pVAX1 (FIG. 16A). The frequencyof CD3⁺CD4⁺TNF-α⁺ T cells was about 6-fold and about 11-fold greater inthe splenocyte populations isolated from mice immunized with theMERS-HCoV-ΔCD and MERS-HCoV-WT constructs, respectively, as compared tothe control pVAX1 (FIG. 16B). The frequency of CD3⁺CD4⁺IL-2⁺ T cells wasabout 12.5-fold and about 30-fold greater in the splenocyte populationsisolated from mice immunized with the MERS-HCoV-ΔCD and MERS-HCoV-WTconstructs, respectively, as compared to the control pVAX1 (FIG. 16C).The frequency of CD3⁺CD4⁺IFN-γ⁺TNF-α⁺ T cells was about 7.5-fold andabout 17.5-fold greater in the splenocyte populations isolated from miceimmunized with the MERS-HCoV-ΔCD and MERS-HCoV-WT constructs,respectively, as compared to the control pVAX1 (FIG. 16D). Accordingly,these data indicated that the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs,unlike the control pVAX1, induced a polyfunctional T cell response, inwhich increased numbers of CD3⁺CD4⁺ T cells produced IFN-γ, TNF-α, IL-2,or both IFN-γ and TNF-α.

FIGS. 17A, 17B, 17C, and 17D show the measured frequency of CD3⁺CD8⁺ Tcells producing IFN-γ, TNF-α, IL-2, or both IFN-γ and TNF-α,respectively, in the stimulated splenocyte populations. The data inFIGS. 17A-17D represented, for each group, splenocytes isolated from 4mice. The data in FIGS. 17A-17D also represented mean±SEM. The frequencyof CD3⁺CD8⁺ T cells producing IFN-γ, TNF-α, IL-2, or both IFN-γ andTNF-α was significantly increased in the MERS-HCoV-WT and MERS-HCoV-ΔCDgroups as compared to the control pVAX1.

In particular, the frequency of CD3⁺CD8⁺IFN-γ⁺ T cells was about 8-foldand about 13-fold greater in the splenoctye populations isolated frommice immunized with the MERS-HCoV-ΔCD and MERS-HCoV-WT constructs,respectively, as compared to the control pVAX1 (FIG. 17A). The frequencyof CD3⁺CD8⁺TNF-α⁺ T cells was about 7-fold greater in the splenocytepopulations isolated from mice immunized with the MERS-HCoV-ΔCD orMERS-HCoV-WT construct as compared to the control pVAX1 (FIG. 17B). Thefrequency of CD3⁺CD8⁺IL-2⁺ T cells was about 2.5-fold greater in thesplenocyte populations isolated from mice immunized with theMERS-HCoV-ΔCD or MERS-HCoV-WT construct as compared to the control pVAX1(FIG. 17C). The frequency of CD3⁺CD8⁺IFN-γ⁺TNF-α⁺ T cells was about50-fold and about 90-fold greater in the splenocyte populations isolatedfrom mice immunized with the MERS-HCoV-ΔCD and MERS-HCoV-WT constructs,respectively, as compared to the control pVAX1 (FIG. 17D). Accordingly,these data indicated that the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs,unlike the control pVAX1, induced a polyfunctional T cell response, inwhich increased numbers of CD3⁺CD8⁺ T cells produced IFN-γ, TNF-α, IL-2,or both IFN-γ and TNF-α.

In summary, the above data indicated that the MERS-HCoV-WT andMERS-HCoV-ΔCD constructs significantly induced polyfunctional CD3⁺CD4⁺and CD3⁺CD8⁺ T cells that produced IFN-γ, TNF-α, IL-2, or both IFN-γ andTNF-α. The MERS-HCoV-WT and MERS-HCoV-ΔCD constructs supported greaterpolyfunctionality of both CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells than the controlpVAX1. Accordingly, the consensus constructs were capable of eliciting apolyfunctional cellular immune response that was reactive to theMERS-CoV spike antigen.

Example 8 Neutralizing Antibodies

As described above in Example 5, the MERS-HCoV-WT and MERS-HCoV-ΔCDconstructs induced a humoral immune response. To further examine theinduced humoral immune response, the level of neutralizing antibodiesassociated with mice immunized with pVAX1, MERS-HCoV-WT construct, orMERS-HCoV-ΔCD construct was examined. Specifically, sera were diluted inminimal essential medium and incubated with 50 μl of DMEM containing 100infectious HCoV-EMC/2012 (Human Coronavirus Erasmus Medical Center/2012)particles per well at 37° C. After 90 minutes, the virus-serum mixturewas added to a monolayer of Vero cells (100,000 cells per well) in a96-well flat bottom plate and incubated for 5 days at 37° C. in a 5% CO₂incubator. The titer of neutralizing antibody for each sample wasreported as the highest dilution with which less than 50% of the cellsshowed CPE. Values were reported as reciprocal dilutions. All thesamples were run in duplicate so that the final result was taken as theaverage of the two samples.

The results are shown below in Table 1. Immunization with theMERS-HCoV-WT and MERS-HCoV-ΔCD constructs, unlike the control pVAX1,induced significant levels of neutralizing antibodies that were reactiveto the MERS-CoV spike antigen.

TABLE 1 Titer of Neutralizing Antibodies. Average Repli- Repli-Reciprocal Mouse Number cate 1 cate 2 Dilutions MERS-HCoV-WT Mouse 1 320320 320 MERS-HCoV-WT Mouse 2 2560 2560 2560 MERS-HCoV-WT Mouse 3 320 640480 MERS-HCoV-WT Mouse 4 480 480 480 MERS-HCoV-WT Mouse 5 1610 1610 1610MERS-HCoV-WT Mouse 6 1280 640 960 MERS-HCoV-WT Mouse 7 320 160 240MERS-HCoV-WT Mouse 8 640 320 480 MERS-HCoV-WT Mouse 9 320 640 480MERS-HCoV-ΔCD Mouse 1 320 160 240 MERS-HCoV-ΔCD Mouse 2 160 80 120MERS-HCoV-ΔCD Mouse 3 320 320 320 MERS-HCoV-ΔCD Mouse 4 640 320 480MERS-HCoV-ΔCD Mouse 5 640 320 480 MERS-HCoV-ΔCD Mouse 6 640 640 640MERS-HCoV-ΔCD Mouse 7 320 160 240 MERS-HCoV-ΔCD Mouse 8 160 160 160MERS-HCoV-ΔCD Mouse 9 1280 640 960 MERS-HCoV-ΔCD Mouse 10 320 320 320pVAX1 Mouse 1 CPE in all wells 0 pVAX1 Mouse 2 CPE in all wells 0 pVAX1Mouse 3 CPE in all wells 0 pVAX1 Mouse 4 CPE in all wells 0 pVAX1 Mouse5 CPE in all wells 0 pVAX1 Mouse 6 CPE in all wells 0 pVAX1 Mouse 7 CPEin all wells 0 pVAX1 Mouse 8 CPE in all wells 0 pVAX1 Mouse 9 CPE in allwells 0 pVAX1 Mouse 10 CPE in all wells 0

In summary, the data provided in the Examples herein demonstrated thatthe MERS-HCoV-WT and MERS-CoV-ΔCD constructs were effective vaccinesthat significantly induced both humoral and cellular immune responsesthat were reactive to the MERS-CoV spike antigen. The induced humoralimmune response included increased titers of IgG antibodies andneutralizing antibodies that were immunoreactive with the MERS-CoV spikeantigen as compared to a construct lacking either consensus antigen(i.e., pVAX1). The induced cellular immune response included increasedCD3⁺CD4⁺ and CD3⁺CD8⁺ T cell responses that produced IFN-γ, TNF-α, IL-2,or both IFN-γ and TNF-α as compared to the construct lacking eitherconsensus antigen (i.e., pVAX1).

Example 9 Materials and Methods for Examples 10-19

Cell Culture, Plasmids, and Expression of MERS-HCoV-Spike Protein.

HEK293T cells (ATCC#: CRL-N268) and Vero-E6 cells (ATCC#: CRL-1586) weregrown in DMEM with 10% FBS (DMEM). The MERS-HCoV-Spike WT and SpikeΔCDplasmid DNA constructs encoded an optimized consensus sequence of theMERS Spike protein (S). In addition, the Ig heavy chain epsilon-1 signalpeptide was fused to the N-terminus of each sequence, replacing theN-terminal methionine, which facilitated expression. Each gene wasgenetically optimized for expression in mice, including codon- andRNA-optimization. The optimized genes were then sub-cloned into modifiedpVax1 mammalian expression vectors under the control of thecytomegalovirus immediate-early (CMV) promoter (GenScript). Constructionof these MERS-HCoV-Spike WT and SpikeΔCD plasmid DNA constructs is alsodescribed above in Examples 1 and 2.

For in vitro expression studies, transfection was carried out usingTurboFectin 8.0 reagent, following the manufacturer's protocols(OriGene). Briefly, cells were grown to 80% confluence in a 35-mm dishand transfected with 3 μg of Spike plasmid. The cells were harvested 48hours (h) post transfection, washed twice with phosphate-buffered saline(PBS), and then suspended in cell lysis buffer (Cell SignalingTechnology) to verify the expression of Spike protein by Westernblotting analysis.

Mice and Immunization.

Female C57BL-6 mice (6-8 weeks old; Jackson Laboratories) were used inthese experiments and divided into three experimental groups. Allanimals were housed in a temperature-controlled, light-cycled facilityin accordance with the guidelines of the National Institutes of Health(Bethesda) and the University of Pennsylvania (Philadelphia, Pa., USA)Institutional Animal Care and Use Committee (IACUC). All immunizationswere delivered into the tibialis anterior (25 μg) in a total volume of25 μl by in vivo minimally invasive EP delivery technologies (MID-EP).

Single-cell suspensions of spleens were prepared from the immunizedmice. Briefly, spleens from freshly euthanized mice were collectedindividually in 10 ml of RPMI 1640 supplemented with 10% FBS (R10), thenprocessed via a paddle blender (STOMACHER 80 paddle blender; A. J.Seward and Co. Ltd., London, England) for 60 seconds on high speed.Processed spleen samples were filtered through 45 μm nylon filters thencentrifuged at 800×g for 10 minutes at room temperature. Cell pelletswere resuspended in 5 ml ACK lysis buffer (Life Technology) for 5minutes at room temperature, and PBS was then added to stop thereaction. Samples were again centrifuged at 800×g for 10 minutes at roomtemperature. Cell pellets were suspended in R10 at a concentration of1×10⁷ cells/ml then passed through a 45 μm nylon filter before use inenzyme-linked immunosorbent spot (ELISpot) assay and flow cytometricanalysis.

ELISpot Analysis.

Antigen specific T cell responses were determined using IFN-γ ELISpot.Briefly, PVDF 96-well plates (Millipore) were coated with purifiedanti-mouse IFN-γ capture antibody and incubated for 24 h at 4° C. (R&DSystems). The following day, plates were washed and blocked for 2 h with1% BSA and 5% Sucrose. Two hundred thousand splenocytes from theimmunized mice were added to each well and stimulated overnight at 37°C. in 5% CO₂ in the presence of RPMI 1640 (negative control), Con A (5ug/mL; positive control), or specific peptide antigens (Ag) (10 μg/ml;GenScript). Peptide pools consisted of 15-mer peptides overlapping by 11amino acids (GenScript). After 24 h of stimulation, the cells werewashed and incubated for 24 h at 4° C. with biotinylated anti-mouseIFN-γ Abs (R&D Systems). The plates were washed, andstreptavidin-alkaline phosphatase (R&D Systems) was added to each welland incubated for 2 h at room temperature. The plates were washed, and5-bromo-4-chloro-3′-indolylphosphate p-toluidine salt and nitro bluetetrazolium chloride (Chromogen color reagent; R&D Systems) were addedto each well. The plates were then rinsed with distilled water and driedat room temperature overnight. Spots were counted by an automatedELISpot reader (CTL Limited).

Flow Cytometry and Intracellular Cytokine Staining (ICCS) Assay.

Splenocytes were added to a 96-well plate (1×10⁶/well) and werestimulated with pooled MERS antigen peptide for 5-6 h at 37 C/5% CO₂ inthe presence of Protein Transport Inhibitor Cocktail (Brefeldin A andMonensin; eBioscience) according to the manufacturer's instructions. TheCell Stimulation Cocktail (plus protein transport inhibitors) (phorbol12-myristate 13-acetate (PMA), ionomycin, brefeldin A and monensin;eBioscience) was used as a positive control and R10 media as negativecontrol. All cells were then stained for surface and intracellularproteins as described by the manufacturer's instructions (BD). Briefly,the cells were washed in FACS buffer (PBS containing 0.1% sodium azideand 1% FCS) before surface staining with flourochrome-conjugatedantibodies. Cells were washed with FACS buffer, fixed and permeabilizedusing the BD Cytofix/Ctyoperm™ (BD) according to the manufacturer'sprotocol followed by intracellular staining. The following antibodieswere used for surface staining: LIVE/DEAD Fixable Violet Dead Cell stainkit (Invitrogen), CD19 (V450; clone 1D3; BD Biosciences) CD4 (FITC;clone RM4-5; ebioscience), CD8 (APC-Cy7; clone 53-6.7; BD Biosciences);CD44 (A700; clone IM7; Biolegend). For intracellular staining, thefollowing antibodies were used: IFN-γ (APC; clone XMG1.2; Biolegend),TNF-α (PE; clone MP6-XT22; ebioscience), CD3 (PerCP/Cy5.5; clone145-2C11; Biolegend); IL-2 (PeCy7; clone JES6-SH4; ebioscience). Alldata was collected using a LSRII flow cytometer (BD Biosciences) andanalyzed using FlowJo software (Tree Star) and SPICE v5. Boolean gatingwas performed using FlowJo software to examine the polyfunctionality ofthe T cells from vaccinated animals.

Immungoen-Specific ELISA.

An enzyme-linked immunosorbent assay (ELISA) was used to determine thetiters of mouse sera. Briefly, 5 μg/ml of purified recombinant humanbetacoronavirus-Spike protein 2c EMC/2012 (clade A) (Sino BiologicalInc.) was used to coat 96-well microtiter plates (Nalgene NuncInternational, Naperville, Ill.) at 4° C. overnight. After blocking with10% FBS in PBS, plates were washed 4 times with 0.05% PBST (Tween20 inPBS). Serum samples from immunized mice were serially diluted in 1% FBS,0.2% PBST, added to the plates, and then incubated for 1 h at roomtemperature. Plates were again washed 4 times in 0.05% PBST, and thenincubated with HRP-conjugated anti-mouse IgG diluted according to themanufacturer's instructions (Sigma Aldrich) for 1 h at room temperature.Bound enzyme was detected by adding OPD (o-Phenylenediaminedihydrochloride) tablets according to the manufacturer's instructions(SIGMAFAST; Sigma Aldrich). The reaction was stopped after 15 minuteswith the addition of 2N H₂SO₄. Plates were then read at an opticaldensity of 450 nm on a 96 Microplate Luminometer (GLOMAX; Promega). Allsamples were plated in duplicate. End-point-titers were determined asthe highest dilution with an absorbance value greater than the meanabsorbance value from at least three normal sera plus three standarddeviations.

Indirect Immunofluorescent Assay (IFA).

For cells transfected with MERS-HCoV-Spike plasmid, slides were fixedwith ice-cold acetone for 5 min. Nonspecific binding was then blockedwith 5% skim milk in PBS at 37° C. for 30 min. The slides were thenwashed in PBS for 5 min and subsequently incubated with sera fromimmunized mice at 1:100 dilutions. Following a 1 h incubation, slideswere washed as described above and incubated with Alexa Fluor 488 goatanti-mouse IgG (Invitrogen) diluted in PBS containing 1 ug/mL and 46-diamidino-2-phenylindole (DAPI) at 37° C. for 30 min. After washingout the unbound antibodies, the coverslips were mounted with ProlongGold antifade reagent (Invitrogen), and the slides were observed under aconfocal microscope (LSM710; Carl Zeiss). The resulting images wereanalyzed using the Zen software (Carl Zeiss).

Preparation of MERS-HCoV-Spike Pseudo Viral Particles.

The codon-optimized consensus MERS-HCoV Spike gene was synthesized andsub-cloned into the pVax1 vector. MERS-HCoV-Spike pseudovirus wereprepared. Specifically, 2×10⁶ HEK293T cells were seeded in 10-cm tissueculture plates and transfected with plasmids expressing MERS-Spike orVSV-G protein using TurboFectin 8.0 (OriGene) transfection reagents atabout 80% confluence. To produce HIV/MERS-Spike pseudoparticles, 10 μgpNL Luc E-R- (NIH-AIDS-Reagent Program) and 10 μg pVax1-MERS-Spike fromvarious clades were co-transfected into cells. After 12 h twelve hours,transfection media was removed and replaced with fresh media forapproximately 12 h and cells were incubated for 24-48 h at 37° C. Thepseudovirion-containing media was collected, filtered, and pseudovirionswere concentrated at 40,000 rpm in a SW41 rotor for 1 h through a 20%sucrose cushion prepared in TNE buffer (10 mM Tris, 135 mM NaCl, 2 mMEDTA, pH 8.0). The pellet was resuspended overnight in 500 μL TNEbuffer, aliquoted and stored at −80° C.

Neutralization Assay.

The 50% tissue culture infectivity dose (TCID₅₀) was calculated and astandard concentration of virus (i.e. 100TCID₅₀) was used for theneutralization test throughout the study, which was performed with themouse sera from MERS-HCoV DNA immunized animals. Briefly, the mouse serawere serially diluted in MEM and incubated with 50 ul of DMEM containing100 infectious HCoV-EMC/2012 (Human Coronavirus Erasmus MedicalCenter/2012) particles per well at 37° C. After 90 min, the virus-serummixture was add to a monolayer of Vero cells (100,000 cells/per well) ina 96-well flat bottom plate and incubated for 5 days at 37° C. in a 5%CO₂ incubator. The titer of neutralizing antibody for each sample wasreported as the highest dilution with which less than 50% of the cellsshow CPE. Values were reported as reciprocal dilutions. All the sampleswere run in duplicate so the final result was taken as the average ofthe two. The percent neutralization was calculated as follows: Percentneutralization={1-PFU mAb of interest (each concentration)/Mean PFUnegative control (all concentrations)}. Neutralization curves weregenerated and analyzed using GraphPad Prism 5. Nonlinear regressionfitting with sigmoidal dose-response (variable slope) was used todetermine the IC₅₀ and IC₈₀. Positive and negative control sera wereincluded to validate the assay.

For the neutralization tests, MERS-Spike pseudoparticles (50 ng) werepre-incubated with serially diluted mouse sera for 30 min at 4° C. andthen added to cells in triplicate. CPE was read at three days postinfection. The highest serum dilution that completely protected thecells from CPE in half of the wells was taken as the neutralizingantibody titer. For the luciferase-based assay, MERS-Spikepseudoparticels infected cells were lysed in 50 μl protein lysis bufferand 100 μl of luciferase substrate at two days post infection.Luciferase activity was measured in a 96 Microplate Luminometer (GLOMAX;Promega Corporation).

Cell Viability Assay and Annexin V/FITC Assay.

Cell viability was determined using3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, 5mg/ml, Sigma). The cultures were initiated in 96-well plates at adensity of 2.5×10³ cells per well. After 48 h incubation, cells wereinfected with MERS-pesudovirus and cultured for 48 h. After incubation,15 μl of MTT reagent was added to each well and incubated for 4 h at 37°C. in the dark. The supernatant was aspirated and formazan crystals weredissolved in 100 μl of DMSO at 37° C. for 15 min with gentle agitation.The absorbance per well was measured at 540 nm using the a LuminometerReader (GLOMAX; Promega). Data was analyzed from three independentexperiments and then normalized to the absorbance of wells containingmedia only (0%) and untreated cells (100%). IC₅₀ values were calculatedfrom sigmoidal dose response curves with Prism GraphPad software.

The Annexin V-FITC binding assay was performed according to themanufacturer's instructions using the Annexin V-FITC detection kit I (BDBiosciences). The cells were infected with MERS-pseudovirus for 12 h.The cells were counted after trypsinization and washed twice with coldPBS. The cell pellet was resuspended in 100 μl of binding buffer at adensity of 1×10³ cells per ml and incubated with 5 μl of FITC-conjugatedAnnexin-V and 5 μl of PI for 15 min at room temperature in the dark.Four hundred microliters of 1× binding buffer was added to each sampletube, and the samples were immediately analyzed by flow cytometer (BDBiosciences). Data were analyzed using FlowJo software (Tree Star).

Non-Human Primate (NHP) Immunization and MERS Challenge.

Three groups of Rhesus macaques received 3 doses (prime and 2 boosts)administered 3 weeks apart (weeks 0, 3, 6). Group 1 and 2 macaquereceived a total of 0.5 mg and 2 mg/dose of a DNA vaccine expressing theMERS-Spike (N=4) vaccine by intramuscular immunizations with the EPdevice. Groups 1 and 2 received the full-length consensus MERS Spikeantigen. Group 3 macaque received a total of 2 mg/dose of a controlvaccine (pVax1). The dose and immunization regimen of DNA vaccine usedin these studies were previously determined to be optimum in rhesusmacaques. Blood was collected after each dose to analyze serum antibody,and neutralization and systemic T cell responses. Animals wereanesthetized intramuscularly with ketamine HCL (10-30 mg/kg). Thevaccine was administered to each thigh (one injection site per thigh pervaccination) and delivered by the intramuscular (IM) route. Immediatelyfollowing the DNA injection, 3 pulses at 0.5 A constant current with 52ms pulse length with is between pulses was applied for IMadministration.

NHP Challenge.

Three groups of Rhesus macaques received 3 doses (prime and 2 boosts)administered 3 weeks apart (weeks 0, 3, 6). Group 1 and 2 macaquereceived a total of 0.5 mg and 2 mg/dose of a DNA vaccine expressing theMERS-Spike (N=4) vaccine by intramuscular immunizations over the withthe EP device. Group in 3 macaque received a total of 2 mg/dose of acontrol vaccine (pVax1). The dose, immunization regimen of DNA vaccineto be used in these studies were previously determined to be optimum inrhesus macaques. Blood was collected after each dose to analyze serumantibody and Neutralization and systemic T cell responses. Animals wereanesthetized intramuscularly with ketamine HCL (10-30 mg/kg). Thevaccine was administered to each thigh (one injection site per thigh pervaccination) and delivered by the IM route. Immediately following theDNA injection, a 3 pulses at 0.5 A constant current with 52 ms pulselength with is between pulses was applied for IM administration.

Challenge: 12 healthy rhesus macaques (Macaca mulatta), aged 4-6 years,were inoculated with a total of 7×10⁶ TCID50 of MERS-CoV by combinedintratracheal, intranasal, oral and ocular routes as previouslyestablished (Falzarano et al Nature Medicine 19, 1313-1317 (2013)Animals were randomly assigned to either the treated or untreated groupin a nonblinded manner.

Statistical Analysis.

The animal experiments to evaluate immune responses were repeated atleast three times and the response of each mouse was counted as anindividual data point for statistical analysis. All data were presentedas means±standard deviations. Data obtained from animal studies andvarious immune assays were examined by using one-way ANOVA fromGraphPad; differences were considered significant at p<0.05. GraphPadPrism v. 5.0 (GraphPad Software, Inc.) was used for statisticalanalysis.

Example 10 Cloning and Expression of MERS-HCoV Spike and Spike ΔCDProteins

The consensus sequence for MERS-HCoV Spike gene was generated from 16Spike genomic sequences deposited in the GenBank-NCBI database. For theimmunogen design, sequences from both Clade A and B were included in theconsensus sequence and the resulting consensus sequence was tested bymeasuring the neutralizing activity across clades A and B viral strainsof MERS-HCoV. As shown in FIG. 19A, the phylogenetic position of theMERS-HCoV-Spike consensus sequence was centered in the Clade B quadrant,which was due to the multiple sequences that fall within this clade.Furthermore, two consensus sequences of the MERS Spike glycoprotein weredesigned, in which one construct's cytoplasmic domain sequence was fullyintact and the second construct's cytoplasmic domain was truncated.These constructs were termed the MERS-HCoV Spike (Spike-Wt) and thecytoplasmic portion truncated (SpikeΔCD), respectively. For bothimmunogens, several modifications were made to enhance in vivoexpression, including addition of a highly efficient IgE leader peptidesequence to facilitate expression and mRNA export. The inserts were thensub-cloned into the pVax1 vector (FIG. 19B). Construction of theseSpike-Wt and SpikeΔCD DNA constructs is also described above in Examples1 and 2.

The plasmids were transfected into 293T cells separately, and theexpression of Spike protein was evaluated by Western blotting. The mouseantiserum specific for Spike-Wt proteins from the immunized mice wereused to detect the expression of Spike protein from the plasmidtransfected cells lysates. At 48 hours post-transfection, proteinlysates were extracted. Strong specific bands of MERS-Spike protein (140kDa) was detected in Spike-Wt and SpikeΔCD transfected cells, but not inlysates from cells transfected with the control vector (an empty pVax1plasmid) (FIG. 19C).

In addition, the expression and localization of Spike protein upontransfection was investigated using an immunoflorescent assay (IFA). TheIFA using mouse Spike antiserum revealed a strong signal was present inthe cytoplasm (FIG. 19D). The positive signal was not detected in intactcells transfected with pVax1 vector. Both full length construct(Spike-Wt) and the cytoplasmic domain mutant construct (SpikeΔCD) werelocalized similarly within transfected cells. These results demonstratethe ability of the MERS-HCoV constructs to express strongly in mammaliancells and that antibodies induced by these constructs can bind theirtarget antigen.

Example 11 Functional Expression and Infection by MERS-HCoV DNAConstructs

To determine the functionality of the consensus sequence immunogens(e.g., viral binding and entry), a pseudoviral expression system wasdeveloped to test the viral entry properties of the consensusconstructs. Specifically, MERS-HCoV pseudoviral particles were producedby co-transfection of 293T cells with plasmids encoding the MERS-Spikeantigen(s) and an HIV-1 luciferase reporter plasmid, which does notexpress HIV-1 envelope (FIG. 20A). Particles were produced bytransfection of 293T cells with various MERS-Spike gene+lentiviralgenome fragment. This generated robust viral particle formation. Tocharacterize MERS-Spike mediated infection, a time course analysis wasperformed measuring the luciferase activity in cells synchronouslyinfected with the consensus MERS-Spike pseudovirus (FIG. 20B). As seenin FIG. 20C, inoculation of Vero cells and A549 cells cell lines thatexpressed functional receptors for MERS-CoV with pseudoviral particlesproduced a strong luciferase signal, indicating productive infectionwith the pseudovirus. These experiments showed the development of MERSpseudovirions with Spike protein, which entered target cells andestablished a pseudovirus-based inhibition assay for the detection ofneutralizing antibodies.

Example 12 Increased Immunogenicity of MERS-HCoV DNA Vaccines in Mice

To investigate the immunogenicity of the consensus and modified Spikeglycoproteins, constructs were analyzed for their ability to elicitimmune responses after intramuscular injection in C57BL/6 mice. FemaleC57BL/6 mice (n=9) were vaccinated with 25 μg of one of three DNAplasmids: Spike-Wt, SpikeΔCD or a control pVax1 vector. Immediatelyfollowing each immunization, the adaptive electroporation (EP) systemwas used. Animals were vaccinated three times at two-week intervals, andimmune responses were measured one week following the third immunizationas described in FIG. 21A.

Cell-mediated immunity was evaluated by using a standard ELISpot assayto monitor the ability of splenocytes from the immunized mice to secretecytokines after antigen specific in vitro restimulation with peptidepools encoding the entire protein region of the MERS-Spike glycoprotein.ELISpot assays were carried out one week following the thirdimmunization. As shown in FIG. 21B, the splenocytes from Spike-Wt aswell as immunized SpikeΔCD vaccination induced strong cellular immuneresponses against multiple peptide pools. Peptides in pools 2 and 5appeared dominant with both constructs.

Based on these T cell responses, detailed mapping against 31 diversepools spanning the entire MERS-Spike protein was performed. Two hundredtwenty seven peptide pools, containing 15-mer peptides with 11 aminoacid overlaps and spanning the residues of Spike protein, weregenerated. Thirty-one peptide pools were prepared using the matrixformat and were tested. Following restimulation with the peptide, astrong CD8⁺ T cell response was detected against several regions on theSpike protein (FIG. 21C and FIG. 21D). There were fifteen matrix poolsshowing more than 100 spots, indicating that MERS-Spike elicited a broadrange of cellular immune responses. Four-peptide-pools in the regionfrom amino acid 301-334 were identified. Importantly the Spike-Wt aswell as Spike-ΔCD immunogen reacted to 4 major regions spanning thepeptide pools 4-6, 11-13, 18-21 and 29-31. However, the dominant poolsappeared to span 18-21. These pools included a computer identified CD8⁺T-lymphocyte immunodominant epitope at amino acids 307-321(RKAWAAFYVYKLQPL (SEQ ID NO:7)), which may be a dominant response byboth antigens (FIGS. 21C and 21D).

Example 13 MERS-Spike Vaccine-Induced T Cell Responses werePolyfunctional

In order to further determine the phenotype of the induced T cellresponses, the polyfunctional T cell responses were assessed.Polychromatic flow cytometry was employed to measure the production ofIFN-γ, IL-2 and TNF-α induced in an antigen specific fashion in bothCD4⁺ and CD8⁺ T cells. Representative flow cytometry profiles ofMERS-Spike-specific IL-2, TNF-α and IFN-γ, secreting CD4⁺ and CD8⁺ Tcells are shown in FIGS. 25A and 25B respectively. The constructsgenerated comparable responses from CD8⁺ T cells; the full-lengthconstruct induced significantly higher percentages of CD4⁺ T cellssecreting IL-2, TNF-α and IFN-γ.

The magnitude of vaccine-induced CD4⁺ and CD8⁺ T cell responses forMERS—HCoV-Spike as well MERS-HCoVΔCD vaccine construct were compared.Using Boolean gating, the ability of individual cells to producemultiple cytokines, i.e. polyfunctionality of the vaccine-induced CD4⁺and CD8⁺ T cell response, was assessed (FIGS. 25C and 25D). Thisanalysis showed that the CD8⁺ T cell responses were similar in the fulllength or truncated Spike protein vaccine groups, however, the magnitudeof the CD4⁺ T cell responses were greater in the full length Spikeantigen vaccine group. Seven distinct Spike-specific CD4⁺ and CD8⁺T-Cell populations were identified. Although the proportion of tri, bi-,and mono-functional cells varied slightly between these two vaccinegroups, there were trends observed in both groups with overall magnitudefavoring the full length construct in both CD4⁺ as well as CD8⁺ T cellresponse induction. When the responses were then further divided intotheir 7 possible functional combinations, it was observed that CD8⁺T-cells in both of the vaccination groups were similar except forinduction of CD8⁺ T cells that produce IFN-γ, which again favored thefull length construct in magnitude (FIGS. 25C and 25D).

Example 14 MERS-HCoV-Spike Vaccination Induced Sufficient BindingAntibody Responses as Well as Neutralizing Antibody (Nab) Responses inMice

The induction of humoral immunity by the two constructs was alsoexamined. Serum samples were obtained before and after DNA immunizationin mice. The anti-Spike humoral immune responses were analyzed forbinding to recombinant Spike antigen as well as in functional antibodystudies. As shown in FIGS. 18A and 22A, all vaccinated animals inducedspecific antibody responses compared to the control animals that wereimmunized with plasmid backbone. Similar to the CD4⁺ T cell responses,the binding antibody activity was stronger in the full-length immunizedanimals quantified as end point titer (FIGS. 18B and 22B). Theantibodies generated from the immunized mice also bound to theMERS-Spike recombinant protein in Western blot assay (FIG. 22C).Collectively, these data indicated that the Spike DNA vaccine inducedspecific antibody production and antibodies that bound specifically tothe target Spike antigen.

Example 15 Antisera to MERS-HCoV-Spike Demonstrated NeutralizationActivity Against MERS Virus

The neutralizing activity of serum from mice immunized with either theSpike-Wt or SpikeΔCD vaccine was assessed via a viral neutralizationassay with the Clade A strain virus, HCoV-EMC/2012 (Human CoronavirusErasmus Medical Center/2012). Sera were collected from mice (n=9 pergroup) vaccinated with either Spike-Wt or SpikeΔCD and the negativecontrol pVax1. As shown in FIGS. 18C and 22D, both vaccines inducedneutralizing antibody titers that were significantly higher than seratiters from mice immunized with a control vector (pVax1) alone(p=0.0018). Similar to the antibody binding assays, though notsignificantly different, the truncated construct appeared to induce alower level of neutralizing responses than the full-length construct.

As the neutralization assay was limited due to the lack of availablefull-length virus at this time, the pseudoparticle neutralization assaydescribed above was utilized to test the ability of the antisera toneutralize MERS-Spike pseudovirus. As shown in FIG. 22E, MERS-Spike-Wtvaccinated mouse (n=4) antisera efficiently inhibited infection of Verocells by MERS-Spike pseudovirus with 50% neutralizing Abs titers rangingfrom 120 to 960 and higher. Further, the above sera were evaluated forneutralizing activity against different clade of MERS-CoV infectionusing MERS-pseudo virus based inhibition assay (See the FIG. 24D). FIG.24D shows detection of neutralizing antibodies of vaccinated seraagainst MERS-CoV infection. Briefly, Macaque's sera, at two week afterthe third immunization, were collected from the vaccinated animals andMERS-CoV pseudovirus-based inhibition assay in Vero cells for bothMERS-Low and high dose groups were performed. Pseudovirus entry wasquantified by luciferase activity at 40 hrs post inoculation.

The results indicated that the neutralizing antibodies from these serashowed broader inhibition as tested by the pseudovirus inhibition assay.Overall, these results were consistent with the results obtained fromthe neutralizing assay using wild-type MERS-HCoV and further illustratedthe cross protective nature of the antibody response induced by thisvaccine.

Example 16 Generation of Multi-Functional T Cells in the PeripheralBlood of Macaques Following MERS-Spike DNA Vaccination

Vaccine efficacy against MERS-challenge was also assessed in thepreclinical non-human primates model. This study is outlined in Table 2below.

TABLE 2 Immu- nization Group 1 Group 2 Group 3 Week-0 Control (pVax1)pMERS-Spike Low pMERS-Spike-High Week-3 Control (pVax1) pMERS-Spike LowpMERS-Spike-High Week-6 Control (pVax1) pMERS-Spike Low pMERS-Spike-HighWeek 6-8 Post Post Post Immunization Immunization Immunization T cell &nAbs T cell & nAbs T cell & nAbs Analysis Analysis Analysis Week-8-9Transfer to NIH- Transfer to NIH- Transfer to NIH- BSL4 Lab BSL4 LabBSL4 Lab Week-10 MERS-Challenge MERS-Challenge MERS-Challenge(intranasal (i.n.)) (i.n.) (i.n.) Week 12 Post Challenge Post ChallengePost Challenge Analysis Analysis Analysis

Three groups (low, high and control) of animals (four Indian Rhesusmacaques per group) were immunized with MERS-Spike vaccine as describedin Example 9 (FIG. 23A). To determine the impact of the MERS-Spikevaccine on T cell responses, an ELISpot assay was used to enumerate theT cells in the blood of vaccinated animals secreting IFN-γ in responseto stimulation with pools of overlapping peptides derived from the fulllength of MERS-Spike protein. After three immunizations, the number ofMERS-Spike specific T cells present in the blood of the vaccinatedanimals and the total vaccine response in low dose groups were between600 and 1,100 SFU/million PBMCs, whereas the high dose group showedbetween 100- and 1500 SFU/million PBMCs except one animal and with mostvaccinated animals having a positive IFN-γ response. Results were shownas stacked group mean responses ±standard error of the mean (FIGS. 23Band 23C). These date indicated that the MERS-Spike vaccine induced aspecific T cell response that was polyfunctional as compared to animalsthat did not receive the vaccine (i.e., the pVax1 control group).

Example 17 Detection of Humoral Immune Response

MERS-spike specific antibodies were detected in serum obtained fromvaccinated animals two weeks after the third immunization. First, theMERS-Spike specific ELISA was performed utilizing full-length MERS-Spikeprotein. Binding ELISA results are shown in FIGS. 24A, 27A, and 27B. Allpre-vaccination (day 0) sera were negative for MERS-Spike specificantibodies by ELISA. After the third vaccination with MERS-Spike, thelow dose and high dose groups showed that high-titer antibodies wereproduced. Significant increases in endpoint MERS-spike specific antibodytiters of greater than 10,000 were observed in both low and high dosevaccine immunized monkeys with MERS-Spike protein (FIG. 24B).Accordingly, animals receiving the vaccine has a MERS-Spike antigenspecific humoral immune response unlike the animals that did not receivethe vaccine (i.e., the pVax1 control group).

Example 18 Increased Capacity to Cross-Neutralizing Antibody Response inNon-Human Primates (NHP)

The neutralizing antibody (Nab) against a lab adapted stock of MERS-HCoVwas also measured from both pre bleed and after the 3rd immunization.Monkeys were immunized three times with either 500 ug or 2 mg of totalDNA by IM-EP and showed high levels of Nabs against live MERS-EMC/2012isolates (Clade A) (FIG. 24C). MERS-CoV genomes were phylogeneticallyclassified into 2 clades, clade A and B clusters. To test this crossclade neutralization activity, cross-neutralization was performedthrough the MERS-pseudovirus that expressed multiple clades of Spikeprotein and elicited neutralizing activity against all other MERS-CoVclades (FIG. 24D). Thus, vaccination with DNA expressing the consensusMERS-Spike led to the development of higher antibody titers and moredurable Nab responses against MERS-CoV. Together, these findingsdemonstrated that the MERS-Spike DNA vaccination generated polyclonalantibodies that not only bound to MERS-Spike protein, but also werefunctionally active, and elicited MERS neutralizing antibodies.

Example 19 MERS Challenge Post-Vaccination and Pathobiology

Experiments were designed to assess clinical observations of rhesusmacaques inoculated with MERS-CoV.

As shown above in Table 2, the NHPs were challenged with MERSintranasally (i.n.) at week 10, which was followed by analysis at week12. This week 12 analysis included x-ray data/pathology analysis.

The control group, which was vaccinated with pVax1 DNA vector, had grosslesions throughout all the lung lobes and significant lesions in thelower lobe at day 5 after the challenge. This gross pathology in thecontrol group was consistent with MERS infection. Table 3 summarizes thepathology of the animals. In summary, the MERS-Spike DNA vaccinatedanimals showed improved clinical parameters with absence of grosslesions as compared to the control group of animals, thereby indicatingthat the MERS-Spike DNA vaccine provided protection against MERSinfection.

TABLE 3 Radiation image findings in lungs of rhesus macaques inoculatedwith MERS-CoV between 1 and 6 dpi. Images and clinical observations weremade on days 1, 3, 5, and 6. Day 1 Day 3 Day 5 Day 6 hCoV50¹Interstitial Diffuse interstitial Diffuse interstitial Serious diffuseinfiltration infiltration present in infiltration present ininterstitial infiltration present in both both Caudal lobes; both Caudallobes; present in both Caudal Caudal lobes Bronchial pattern Bronchialpattern lobes; Bronchial present in right middle present in rightpattern present in right lobe middle lobe middle lobe hCoV51¹Interstitial Diffuse interstitial Diffuse interstitial Diffuseinterstitial infiltration infiltration present in infiltration presentin infiltration present in present in both both Caudal lobes both Caudallobes both Caudal lobes and Caudal lobes and right middle right middlelobe lobe hCoV52¹ Normal Interstitial infiltration Interstitialinfiltration Interstitial infiltration present in both Caudal present inboth present in both Caudal lobes; Small mass Caudal lobes; Small lobes;Small mass in present in mass in right Caudal right Caudal lobe; rightCaudal lobe; Bronchial Bronchial pattern lobe pattern present in presentin both Caudal both Caudal lobes lobes hCoV53¹ Interstitial infiltrationInterstitial infiltration Interstitial infiltration Interstitialinfiltration present in both present in both Caudal present in bothpresent in both Caudal Caudal lobes; Air lobes; Air Caudal lobes; Airlobes; Air Bronchograms Bronchograms Bronchograms Bronchograms observedin right observed in left observed in left observed in left middle lobecaudal, right caudal caudal, right caudal caudal, right caudal andmiddle lobes and middle lobes and middle lobes hCoV54² Normal NormalNormal Normal hCoV55² Interstitial infiltration Interstitialinfiltration Normal Normal present in left caudal, present in leftcaudal, right caudal and right caudal and middle lobes middle lobeshCoV56² Normal Normal Normal Normal hCoV57² Interstitial infiltrationInterstitial infiltration Normal Normal present in left caudal, presentin left Caudal, right caudal and right Caudal and middle lobes middlelobes; Air Bronchograms observed in left caudal, right caudal and middlelobes hCoV58³ Normal Normal Normal Normal hCoV59³ Normal Normal NormalNormal hCoV60³ Normal Normal Normal Normal hCoV61³ Normal Normal NormalNormal ¹Vaccinated with pVax1. ²Vaccinated with MERS-Spike (high).³Vaccinated with MERS-Spike (low)

6. CLAUSES

Clause 1. A vaccine comprising a nucleic acid molecule, wherein: (a) thenucleic acid molecule comprises a nucleic acid sequence having at leastabout 90% identity over an entire length of the nucleic acid sequenceset forth in SEQ ID NO:1; or (b) the nucleic acid molecule comprises anucleic acid sequence having at least about 90% identity over an entirelength of the nucleic acid sequence set forth in SEQ ID NO:3.

Clause 2. The vaccine of clause 1, wherein the nucleic acid moleculecomprises the nucleic acid sequence set forth in SEQ ID NO:1.

Clause 3. The vaccine of clause 1, wherein the nucleic acid moleculecomprises the nucleic acid sequence set forth in SEQ ID NO:3.

Clause 4. The vaccine of clause 1, further comprising: (a) a peptidecomprising an amino acid sequence having at least about 90% identityover an entire length of the amino acid sequence set forth in SEQ IDNO:2; or (b) a peptide comprising an amino acid sequence having at leastabout 90% identity over an entire length of the amino acid sequence setforth in SEQ ID NO:4.

Clause 5. The vaccine of clause 1, wherein the nucleic acid moleculecomprises an expression vector.

Clause 6. The vaccine of clause 1, wherein the nucleic acid molecule isincorporated into a viral particle.

Clause 7. The vaccine of clause 1, further comprising a pharmaceuticallyacceptable excipient.

Clause 8. The vaccine of clause 1, further comprising an adjuvant.

Clause 9. A vaccine comprising a nucleic acid molecule, wherein: (a) thenucleic acid molecule encodes a peptide comprising an amino acidsequence having at least about 90% identity over an entire length of theamino acid sequence set forth in SEQ ID NO:2; or (b) the nucleic acidmolecule encodes a peptide comprising an amino acid sequence having atleast about 90% identity over an entire length of the amino acidsequence set forth in SEQ ID NO:4.

Clause 10. The vaccine of clause 9, wherein the nucleic acid moleculeencodes the peptide comprising the amino acid sequence set forth in SEQID NO:2.

Clause 11. The vaccine of clause 9, wherein the nucleic acid moleculeencodes the peptide comprising the amino acid sequence set forth in SEQID NO:4.

Clause 12. The vaccine of clause 9, further comprising: (a) a peptidecomprising an amino acid sequence having at least about 90% identityover an entire length of the amino acid sequence set forth in SEQ IDNO:2; or (b) a peptide comprising an amino acid sequence having at leastabout 90% identity over an entire length of the amino acid sequence setforth in SEQ ID NO:4.

Clause 13. The vaccine of clause 9, wherein the nucleic acid moleculecomprises an expression vector.

Clause 14. The vaccine of clause 9, wherein the nucleic acid molecule isincorporated into a viral particle.

Clause 15. The vaccine of clause 9, further comprising apharmaceutically acceptable excipient.

Clause 16. The vaccine of clause 9, further comprising an adjuvant.

Clause 17. A nucleic acid molecule comprising the nucleic acid sequenceset forth in SEQ ID NO:1.

Clause 18. A nucleic acid molecule comprising the nucleic acid sequenceset forth in SEQ ID NO:3.

Clause 19. A peptide comprising the amino acid sequence set forth in SEQID NO:2.

Clause 20. A peptide comprising the amino acid sequence set forth in SEQID NO:4.

Clause 21. A vaccine comprising an antigen, wherein the antigen isencoded by SEQ ID NO:1 or SEQ ID NO:3.

Clause 22. The vaccine of clause 21, wherein the antigen is encoded bySEQ ID NO:1.

Clause 23. The vaccine of clause 21, wherein the antigen is encoded bySEQ ID NO:3.

Clause 24. The vaccine of clause 21, wherein the antigen comprises theamino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.

Clause 25. The vaccine of clause 24, wherein the antigen comprises theamino acid sequence set forth in SEQ ID NO:2.

Clause 26. The vaccine of clause 24, wherein the antigen comprises theamino acid sequence set forth in SEQ ID NO:4.

Clause 27. A vaccine comprising a peptide, wherein (a) the peptidecomprises an amino acid sequence having at least about 90% identity overan entire length of the amino acid sequence set forth in SEQ ID NO:2; or(b) the peptide comprises an amino acid sequence having at least about90% identity over an entire length of the amino acid sequence set forthin SEQ ID NO:4.

Clause 28. The vaccine of clause 27, wherein the peptide comprises theamino acid sequence set forth in SEQ ID NO:2.

Clause 29. The vaccine of clause 27, wherein the peptide comprises theamino acid sequence set forth in SEQ ID NO:4.

Clause 30. A method of inducing an immune response against a Middle EastRespiratory Syndrome coronavirus (MERS-CoV) in a subject in needthereof, the method comprising administering a vaccine of clause 1, 9,21, or 27 to the subject.

Clause 31. The method of clause 30, wherein administering includes atleast one of electroporation and injection.

Clause 32. A method of protecting a subject in need thereof frominfection with a Middle East Respiratory Syndrome coronavirus(MERS-CoV), the method comprising administering a vaccine of clause 1,9, 21, or 27 to the subject.

Clause 33. The method of the clause 32, wherein administering includesat least one of electroporation and injection.

Clause 34. A method of treating a subject in need thereof against MiddleEast Respiratory Syndrome coronavirus (MERS-CoV), the method comprisingadministering a vaccine of clause 1, 9, 21, or 27 to the subject,wherein the subject is thereby resistant to one or more MERS-CoVstrains.

Clause 35. The method of clause 34, wherein administering includes atleast one of electroporation and injection.

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art. Such changes and modifications,including without limitation those relating to the chemical structures,substituents, derivatives, intermediates, syntheses, compositions,formulations, or methods of use of the invention, may be made withoutdeparting from the spirit and scope thereof.

1. An immunogenic composition comprising a nucleic acid moleculeselected from the group consisting of: (a) a nucleic acid moleculecomprising an immunogenic fragment of SEQ ID NO:1, wherein the fragmentcomprises at least 60% of SEQ ID NO:1; (b) a nucleic acid moleculecomprising an immunogenic fragment of SEQ ID NO:3, wherein the fragmentcomprises at least 60% of SEQ ID NO:3; (c) a nucleic acid moleculeencoding an immunogenic fragment of SEQ ID NO:2, wherein the fragmentcomprises at least 60% of SEQ ID NO:2; and (d) a nucleic acid moleculeencoding an immunogenic fragment of SEQ ID NO:4, wherein the fragmentcomprises at least 60% of SEQ ID NO:4.
 2. The immunogenic composition ofclaim 1, wherein the nucleic acid molecule comprises an immunogenicfragment of SEQ ID NO:1 or SEQ ID NO:3, wherein the fragment comprisesat least 90% of SEQ ID NO:1 or SEQ ID NO:3.
 3. (canceled)
 4. Theimmunogenic composition of claim 1, further comprising: (a) a peptidecomprising an amino acid sequence having at least about 90% identityover an entire length of the amino acid sequence set forth in SEQ IDNO:2; or (b) a peptide comprising an amino acid sequence having at leastabout 90% identity over an entire length of the amino acid sequence setforth in SEQ ID NO:4.
 5. The immunogenic composition of claim 1, whereinthe nucleic acid molecule comprises an expression vector.
 6. Theimmunogenic composition of claim 1, wherein the nucleic acid molecule isincorporated into a viral particle.
 7. The immunogenic composition ofclaim 1, further comprising a pharmaceutically acceptable excipient. 8.The immunogenic composition of claim 1, further comprising an adjuvant.9. (canceled)
 10. The immunogenic composition of claim 1, wherein thenucleic acid molecule encodes an immunogenic fragment of SEQ ID NO:2 orSEQ ID NO:4, wherein the fragment comprises at least 90% of SEQ ID NO:2or SEQ ID NO:4. 11.-20. (canceled)
 21. An immunogenic compositioncomprising an antigen, wherein the antigen is encoded by an immunogenicfragment of SEQ ID NO:1 or SEQ ID NO:3, wherein the fragment comprisesat least 60% of SEQ ID NO:1 or SEQ ID NO:3. 22.-23. (canceled)
 24. Theimmunogenic composition of claim 21, wherein the antigen comprises animmunogenic fragment of SEQ ID NO:2 or SEQ ID NO:4, wherein the fragmentcomprises at least 90% of SEQ ID NO:2 or SEQ ID NO:4. 25.-26. (canceled)27. An immunogenic composition comprising a peptide, selected from thegroup consisting of: (a) an immunogenic fragment of SEQ ID NO:2, whereinthe fragment comprises at least 60% of SEQ ID NO:2, and (b) animmunogenic fragment of SEQ ID NO:4, wherein the fragment comprises atleast 60% of SEQ ID NO:4 (d).
 28. The immunogenic composition of claim27, wherein the peptide comprises an immunogenic fragment of SEQ ID NO:2or SEQ ID NO:4, wherein the fragment comprises at least 90% of SEQ IDNO:2 or SEQ ID NO:4.
 29. (canceled)
 30. A method of inducing an immuneresponse against a Middle East Respiratory Syndrome coronavirus(MERS-CoV) in a subject in need thereof, the method comprisingadministering an immunogenic composition of claim 1 to the subject. 31.The method of claim 30, wherein administering includes at least one ofelectroporation and injection.
 32. A method of protecting a subject inneed thereof from infection with a Middle East Respiratory Syndromecoronavirus (MERS-CoV), the method comprising administering animmunogenic composition of claim 1 to the subject.
 33. The method of theclaim 32, wherein administering includes at least one of electroporationand injection.
 34. A method of treating a subject in need thereofagainst Middle East Respiratory Syndrome coronavirus (MERS-CoV), themethod comprising administering an immunogenic composition of claim 1 tothe subject, wherein the subject is thereby resistant to one or moreMERS-CoV strains.
 35. The method of claim 34, wherein administeringincludes at least one of electroporation and injection.